Development of a fast DNA-DNA hybridization method based on melting profiles in microplates

DNA-DNA hybridization is still the “gold standard” for the genotypic delineation of bacterial species. However, it is not widely used because traditional DNA-DNA hybridization techniques are rather time-consuming and not easy to perform in routine laboratories. In the present study, DNA of reference strains was digested with Sau3A, ligated with linker oligonucleotides S1/2 and in vitro amplified. The amplified DNA fragments were immobilized on MaxiSorb 96-well plates. DNA isolated from target strains was also digested with Sau3A, ligated with linker oligonuleotides P1/2 and in vitro amplified in the presence of digoxygenin modified dUTP. The labeled amplificate was hybridized to the immobilized reference DNA under isothermal conditions. Thermal denaturation curves of the DNA-DNA hybrids were obtained by using washing solutions of increasing stringency. Remaining hybrids were colorimetrically detected with anti-digoxygenin-horseradish peroxidase anti-bodies. The new method was validated with strains of the genus Pedioccocus for which DNA-DNA similarities have also been determined by the filter hybridization method. In addition, DNA-DNA hybridizations were performed with genotypically defined Enterobacter species.     

Application of a colorimetric DNA-DNA hybridization method for identification ofAeromonas genomic species,isolated from diarrheal stools

The colorimetric DNA-DNA hybridization method for the identification of 18 strains ofAeromonas spp. isolated from human stools was used. Bacterial isolates were also examined by phenotypic characteristics. On the basis of biochemical tests 13 strains were included in phenogroupA. caviœ and 5 strains inA. sobria. Identification to the species level was obtained by colorimetric hybridization method. DNA-DNA similarity values showed that isolates ofA. caviœ group belong to hybridization group (HG) 4 whereas isolates ofA. sobria belong to HG 8/10. DNA relatedness results obtained by the colorimetric method showed good agreement with values detected by the spectrophotometric method. The background in the colorimetric method is lower than in the spectrophotometric one. Results of this study indicate the usefulness of the colorimetric DNA-DNA hybridization in microplates method for the identification ofAeromonas genomic species, isolated from human diarrheal stools.     

A modified method of quantitative colorimetric DNA-DNA hybridization on membrane filters for bacterial identification

Based on conventional membrane filter dot hybridization protocols, a modified method for the quantitative measurement of DNA-DNA reassociation is described. Labeled DNA probes are prepared with Photobiotin and hybridized with immobilized target DNAs on nitrocellulose filters. The extent of hybridization is detected by an enzyme linked assay in microtiter plates using streptavidine-alkaline phosphatase conjugates as reporter enzymes and nitrophenylphosphate as the colorimetric substrate. The procedure is non-destructive and allows the re-use of the filter holding the target DNAs. The results of the membrane filter hybridizations have been compared to spectroscopic DNA-DNA hybridizations and the limits and the applicability of the method for bacterial taxonomy and bacterial identification are discussed.     

A New Enzyme,d-Fructose Dehydrogenase

Genetic relatedness of 14 yeast strains and 2 mold strains was studied by the DNA-DNA hybridization method. The hybridization was performed between mitochondrial-DNA-free, ³²p-labeled DNA of Saccharomyces cerevisiae IAM 4009 and cold DNA of other strains. The DNA homology indices deviated considerably even among S. cerevisiae strains having similar GC contents, but, in general, yeast strains known to be able to mate with S. cerevisiae, showed high homology indices (35∽70%). Other species of Saccharomycetaceae and 6 asporogenous yeast strains exhibited values of 10∽20%. The relatedness suggested from these results was confirmed by the competition experiments and also by the hybridization with ³²P-DNA of Candida pulcherrima IFO 0561. DNA’s of Aspergillus oryzae I and Neurospora crassa IFO 6067 also exhibited low but appreciable homology indices (5∽7%). These results were discussed from the aspects of phylogenetics and also of gene conservation in microorganisms.     

Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.     

Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors

Summary We compared the single-copy DNA sequences of the tetraploid tobacco plant, Nicotiana tabacum, with those of its diploid progenitors N. sylvestris and N. tomentosiformis. We observed that 65% of N. sylvestris and N. tomentosiformis single-copy DNA fragments reacted with each other using moderately stringent hybridization conditions (60° C, 0.18 M Na⁺). An additional 10% sequence homology was detected when the hybridization temperature was reduced by 10° C. The thermal stability of interspecific single-copy DNA duplexes indicated that they were approximately 6% more mispaired than homologous single-copy DNA duplexes. In contrast, we observed almost no single-copy DNA divergence between N. tabacum and its diploid progenitors. Greater than 99% of N. sylvestris and N. tomentosiformis single-copy DNAs reacted with N. tabacum DNA using moderately stringent hybridization conditions. The thermal stability of these duplexes indicated that they contained no more sequence mismatch than homologous single-copy duplexes. Together, our results show that significant single-copy DNA sequence divergence has occurred between the diploid N. sylvestris and N. tomentosiformis genomes. However, by applying our experimental criteria these single-copy DNAs are indistinguishable from their counterparts in the hybrid N. tabacum nucleus.     

Comparison of different cationic probes for electron microscopic visualization of bacterial polyanionic capsular material

We have isolated from the ovine rumen eight bacterial strains belonging to the speciesButyrivibrio fibrisolvens. DNA hybridization studies showed that the eight strains could be divided into four homology groups, of which none was closely related to the type strain ATCC 19171. Measurement of cross-hybridization between selected pairs of bacterial strains showed that DNA types which produced low, but significant, cross-hybridization on dot-blots were able to form heteroduplexes with between 8.4% and 32.9% of the efficiency of homoduplex formation. Thermal denaturation of the same heteroduplexes resulted in Tm values 6.4–7.5°C lower than those of the homologous duplexes formed under the same conditions. In some cases, hybridization between strains was below the level of reliable measurement. Similar experiments with ten recently isolated strains ofBacteroides ruminicola sub-sp.brevis revealed a similar degree of genetic divergence between isolates.     

Deoxyribonucleic acid hybridization betweenSpiroplasma citri and the corn stunt spiroplasma

The DNA base composition of the R8-A2 strain ofSpiroplasma citri and the I-747 and E strains of corn stunt spiroplasma was determined, by using the thermal denaturation temperature (T _m ), to be 26.8, 26.3, and 26.0 mol% (G+C), respectively. By the simple hybridization method, a measurement of the relative binding of homologous or heterologous labeled DNA to unlabeled, DNA-immobilized, nitrocellulose membrane, a homology of 56–60% was demonstrated betweenS. citri and two strains of corn stunt spiroplasma. A relatively higher DNA homology (66–71% between these two organisms was obtained in teh competition experiment, a measurement of the relative ability of homologous or heterologous competitor DNA to block the formation of homologous DNA-DNA duplex.     

Relationship between genome similarity and DNA-DNA hybridization among closely related bacteria

DNA-DNA hybridization has been established as an important technology in bacterial species taxonomy and phylogenetic analysis. In this study, we analyzed how the efficiency with which the genomic DNA from one species hybridizes to the genomic DNA of another species (DNA-DNA hybridization) in microarray analysis relates to the similarity between two genomes. We found that the predicted DNA-DNA hybridization based on genome sequence similarity correlated well with the experimentally determined microarray hybridization. Between closely related strains, significant numbers of highly divergent genes (<55% identity) and/or the accumulation of mismatches between conserved genes lowered the DNA-DNA hybridization signal, and this reduced the hybridization signals to below 70% for even bacterial strains with over 97% 16S rRNA gene identity. In addition, our results also suggest that a DNA-DNA hybridization signal intensity of over 40% indicates that two genomes at least shared 30% conserved genes (>60% gene identity). This study may expand our knowledge of DNA-DNA hybridization based on genomic sequence similarity comparison and further provide insights for bacterial phylogeny analyses.     

Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

A large number of commensal bacteria inhabit the intestinal tract, and interbacterial communication among gut microbiota is thought to occur. In order to analyze symbiotic relationships between probiotic strains and the gut microbiota, a ring with a membrane filter fitted to the bottom was used for in vitro investigations. Test strains comprising probiotic nitto strains (Lactobacillus acidophilus NT and Bifidobacterium longum NT) and type strains (L. acidophilus JCM1132^T and B. longum JCM1217^T) were obtained from diluted fecal samples using the membrane filter to simulate interbacterial communication. Bifidobacterium spp., Streptococcus pasteurianus, Collinsella aerofaciens, and Clostridium spp. were the most abundant gut bacteria detected before coculture with the test strains. Results of the coculture experiments indicated that the test strains significantly promote the growth of Ruminococcus gnavus, Ruminococcus torques, and Veillonella spp. and inhibit the growth of Sutterella wadsworthensis. Differences in the relative abundances of gut bacterial strains were furthermore observed after coculture of the fecal samples with each test strain. Bifidobacterium spp., which was detected as the dominant strain in the fecal samples, was found to be unaffected by coculture with the test strains. In the present study, interbacterial communication using bacterial metabolites between the test strains and the gut microbiota was demonstrated by the coculture technique. The detailed mechanisms and effects of the complex interbacterial communications that occur among the gut microbiota are, however, still unclear. Further investigation of these relationships by coculture of several fecal samples with probiotic strains is urgently required.     

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique.     

<Emphasis Type="Italic">Thalassospira permensis</Emphasis> sp. nov., a new terrestrial halotolerant bacterium isolated from a naphthalene-utilizing microbial consortium

A halotolerant bacterium, strain SMB34^T, was isolated from a naphthalene-utilizing bacterial consortium obtained from primitive technogeneous soil (Verkhnekamsk salt deposit, Perm region, Russia) by enrichment procedure. The strain itself was unable to degrade naphthalene and grew at NaCl concentrations up to 11% (w/v). The 16S rRNA-based phylogenetic analysis showed that the strain belongs to the genus Thalassospira. The DNA-DNA hybridization values between SMB34^T and the type strains of phylogeneti-cally closest species (T. xiamenensis, T. profundimaris and T. tepidiphila) did not exceed 50%. The novel strain could be distinguished from the above species by the cell motility, MALDI/TOF mass spectra of whole cells and a range of physiological and biochemical characteristics. SMB34^T also considerably differs from the recently described species T. xianhensis, with the most striking differences in the DNA G + C content (53. ± 1.0 vs. 61.2 ± 1.0 mol %) and predominant ubiquinones (Q-10 vs. Q-9). The data obtained suggest strain SMB34^T (=VKM B-2527^T = NBRC 106175^T), designated as the type strain, represents a novel species, named Thalassospira permensis sp. nov.     

Biogenesis of mitochondria: XLI. Physical mapping of mitochondrial genetic markers in yeast

This paper describes the physical mapping of five antibiotic resistance markers on the mitochondrial genome of Saccharomyces cerevisiae. The physical separations between markers were derived from studies involving a series of stable spontaneous petite strains which were isolated and characterized for the loss or retention of combinations of the five resistance markers. DNA-DNA hybridization using ³²P-labelled grande mitochondrial DNA was employed to determine the fraction of grande mitochondrial DNA sequences retained by each of the defined petite strains.One petite clone retaining four of the markers in a segment comprising 36% of the grande genome was then chosen as a reference petite. The sequence homology between the mitochondrial DNA of this petite and that of the other petites was measured by DNA-DNA hybridization. For each petite, the total length of its genome derived by hybridization with grande mitochondrial DNA and the fraction of the grande genome retained in common with the reference petite, together with the genetic markers retained in common, were used to position the DNA segment of each petite relative to the reference petite genome. At the same time the relative physical location of the five markers on a circular genome was established. On the basis of the grande mitochondrial genome being defined as 100 units of DNA, the positions of the markers were determined to bo as follows, measuring from one end of the reference petite genome. chloramphenicol (cap1) ~ 0 units erythromycin (ery1) 0 to 15 units oligomycin (oli1) 18 to 19 units mikamycin (mik1) 22 to 25 units paromomycin (par1) 61 to 73 unitsThe general problems of mapping mitochondrial genetic markers by hybridizations involving petite mitochondrial DNA are discussed. Two very important features of petite genomes which could invalidate the interpretation of DNA-DNA hybridization experiments between petite mitochondrial DNAs are the possible presence in the reference petite of differentially amplified DNA sequences, and/or “new” sequences which are not present in the parent grande genome. A general procedure, which overcomes errors of interpretation arising from these two features is described.     

A measurement of the fraction of chloroplast DNA transcribed in Euglena

The fraction of the chloroplast DNA transcribed in the single celled alga $\text{Euglena}$ has been determined by RNA-DNA hybridization. A vast excess of total cell RNA from cells which were rapidly dividing in the light was hybridized in liquid to [¹²⁵I] — chloroplast DNA, and the resulting duplexes separated on hydroxyapatite columns. The contribution of DNA-DNA duplex formation was determined separately and was used to calculate that portion of the duplex which was actually a RNA-DNA hybrid. Sixteen percent of the single stranded chloroplast DNA forms a duplex with this RNA suggesting that 32 percent of the double stranded DNA molecule is being transcribed into RNA under these conditions of cell growth.     

Deoxyribonucleic acid relatedness among strains of the moderately halophilic speciesDeleya halophila

The genomic relatedness among 16 strains assigned to the moderately halophilic speciesDeleya halophila and other 20 representative strains of halophilic and nonhalophilic species was estimated by determination of deoxyribonucleic acid (DNA) base composition and by DNA-DNA hybridization studies. The guanine-plus-cytosine (G + C) base contents, determined from the melting temperature of DNAs ofD. halophila strains, were 66.0–68.8 mol %. DNA-DNA homology studies, determined by membrane filter technique, indicate that the 16 strains ofD. halophila comprise a genetically homogeneous group. High homology (70–100%) was obtained between the type strainD. halophila CCM 3662 and the otherD. halophila strains studied; however, very low DNA relatedness was found between the representative strains ofD. halophila and otherDeleya species (13-0%), as well as other moderately halophilic, marine, or nonhalophilic bacteria investigated.     

DNA-DNA hybridization of some root-nodule bacteria indigenous in the salt-affected soils of egypt

The DNA-DNA hybridization was used to characterize thirty isolates of root-nodule bacteria indigenous to the salt-affected soils of Egypt. Total DNA from different bacterial isolates lacked homology with total DNA probes of the effective strains ofRhizobium leguminosarum andR. meliloti. It is suggested that the genomic structure of the root-nodule bacteria may be modified by salt stress and/or that the effective strains of these bacteria are to be eliminated from the salt-affected soil.     

DNA restriction endonuclease cleavage patterns,DNA sequence similarity and phenotypical characteristics in some strains of Lactobacillus helveticus and Lactobacillus jugurti

Physiological characteristics, deoxyribonucleic acid (DNA) base composition (% guanine + cytosine; % GC), DNA sequence similarity (% DNA-DNA hybridization) and DNA restriction endonuclease cleavage patterns of two strains of Lactobacillus helveticus and four strains of Lactobacillus jugurti were examined.All the strains investigated were closely related genetically, having DNA-DNA hybridization values ranging from 89–100%. Nevertheless, these strains can be differentiated from one another on the basis of the digestion of their DNA by specific restriction endonucleases, such as Bam HI, Eco RI and Hind III. The DNA of these strains shows clear, reproducible and distinct cleavage patterns. Cleavage patterns of DNA from strains L. jugurti S.35.19 and S.36.2 were found to be similar. These findings suggest that fingerprinting of DNA by restriction endonuclease cleavage might provide, in addition to the conventional methods, a useful tool for the characterization of closely related microorganisms at the strain level.     

<Emphasis Type="Italic">Acinetobacter oleivorans</Emphasis> sp. nov. Is capable of adhering to and growing on diesel-oil

A diesel-oil and n-hexadecane-degrading novel bacterial strain, designated DR1^T, was isolated from a rice paddy in Deok-So, South Korea. The strain DR1^T cells were Gram-negative, aerobic coccobacilli, and grew at 20–37°C with the optimal temperature of 30°C, and an optimal pH of 6–8. Interestingly, strain DR1^T was highly motile (swimming and swarming motility) using its fimbriae, and generated N-acyl homoserine lactones as quorum-sensing signals. The predominant respiratory quinone as identified as ubiquinone-9 (Q-9) and DNA G+C content was 41.4 mol%. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species A. calcoaceticus, A. haemolyticus, A. baumannii, A. baylyi, and A. beijerinckii, with which it evidenced sequence similarities of 98.2%, 97.4%, 97.2%, 97.1%, and 97.0%, respectively. DNA-DNA hybridization values between strain DR1^T and other Acinetobacter spp. were all less than 20%. The physiological and taxonomic characteristics with the DNA-DNA hybridization data supported the identification of strain DR1^T in the genus Acinetobacter as a novel species, for which the name Acinetobacter oleivorans sp. nov. is proposed. The type strain is DR1^T (=KCTC 23045^T =JCM 16667^T).     

Enterococcus seriolicida Is a Junior Synonym of Lactococcus garvieae,a Causative Agent of Septicemia and Meningoencephalitis in Fish

The reference strains of Enterococcus seriolicida (ATCC 49156^T) (T = type strain) and of Lactococcus garvieae (ATCC 43921^T) and 30 field strains of Gram-positive cocci isolated from diseased rainbow trout in Italy were found to be phenotypically (API 20 STREPT and API 50 CH) and genetically (DNA-DNA hybridization) similar. The high DNA-DNA homologies (70–100%) and the low ΔT_m(e) (less than 1.1°C) among these strains showed that Enterococcus seriolicida and Lactococcus garvieae are synonyms, describing a single bacterial species. E. seriolicida strains should be classified as L. garvieae, which must be considered as a major pathogen of freshwater and salt water fish with a world-wide distribution.     

Quantitative micro-assay for RNA/DNA hybrids in the study of nucleolar RNA from Chironomus tentans salivary gland cells

A microassay for RNA/DNA hybrids has been designed for the study of RNA from different nuclear components of Chironomus tentans salivary gland cells. The procedure comprises a scale reduction of the conventional filter method for hybridization, using ultraviolet microphotometry for quantitation of RNA and DNA. Hybridization is performed in 0.3 μl of 2 × SSC containing 1–2 × 10^-2 μg DNA, immobilized on a 0.2 mm² ‘micro-filter’, and 0.5–5 × 10⁻² μg RNA, with a specific activity of more than 10⁶ cpm/μg. Results obtained by the microtechnique are found to agree with results obtained by a large-scale, standard procedure. The applicability of the microtechnique is demonstrated in saturation and presaturation-competition experiments. RNA from micro-isolated nucleoli hybridizes a maximum of 0.22% of Chironomus tentans DNA, which corresponds to about 100 cistrons for the 38S ribosomal precursor in the haploid genome. The hybrids show a steep thermal dissociation profile with a T_m of 79 °C, close to the value expected for hybrids with a G + C content of 42%. Presaturation of filter-bound DNA by total unlabelled nucleolar RNA prevents 80% of the subsequent hybridization by labeled nucleolar Presaturation by RNA from one of the two nucleolar organizers prevents to a similar degree the subsequent hybridization by RNA from the other nucleolar organizer. This result indicates a sequence similarity of RNA transcribed in different nucleolar organizers. Further applications of the microtechnique are presented in the accompanying paper where the hybridization properties of chromosomal and nuclear sap RNA are investigated.