Deoxyribonucleic acid homologies among staphylococci: Coagulase-positive reference strains

DNA hybridization results confirm the proposed separation of coagulase-positive staphylococci into two distinct species. Strains ofStaphylococcus aureus representing the various biotypes and different phage typing groups of the human biotype gave high values of reassociation with DNA fromS. aureus reference strain RN 450, at both optimal and restrictive reassociation temperatures. Similar results were obtained between strains ofS. intermedius and its reference strain K 3. Interspecific reassociation between the two coagulase-positive species was low, and each reference strain showed low DNA sequence homology with 10 coagulase-negative species.S. staphylolyticus, strain PS 73, and putative pleiotropic mutants ofS. aureus were shown to be unrelated toS. aureus.     

Saccharomyces barnetti andSaccharomyces spencerorum: two new species ofSaccharomyces sensu lato (van der Walt)

In the course of a study of DNA base sequence homology, 19 strains labelled asSaccharomyces exiguus, its imperfect state,Candida holmii, orC. milleri were examined. Results confirmed the separation ofC. milleri as a separate species. The remaining strains can be divided into three distinct groups of genomic relatedness. The type cultures ofS. exiguus andC. holmii form a cluster with 10 other strains showing variable reassociation values ranging from 100 to 40%. The remaining four strains comprise two separate species showing no nucleotide relatedness between themselves nor to either theS. exiguus complex or toC. milleri. Physiological analyses demonstrate that it is possible to separate these four taxa on the basis of a few simple tests. The two new species are described respectively asSaccharomyces barnetti honoring James Barnett in recognition of his invaluable work in the field of yeast taxonomy andSaccharomyces spencerorum in honor of J.F.T. and Dorothy M. Spencer who have made innumerable contributions to the genetics and biotechnological application of yeasts.     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.     

Polynucleotide Sequence Divergence Among Strains of Escherichia coli and Closely Related Organisms

Polynucleotide sequence similarity tests were carried out to determine the extent of divergence present in a number of Escherichia coli strains, obtained from diverse human, animal, and laboratory sources, and closely related strains of Shigella, Salmonella, and the Alkalescens-Dispar group. At 60 C, relative reassociation of deoxyribonucleic acid (DNA) from the various strains with E. coli K-12 DNA ranged from 100 to 36%, with the highest level of reassociation found for three strains derived from K-12, and the lowest levels for two “atypical” E. coli strains and S. typhimurium. The change in thermal elution midpoint, which indicates the stability of DNA duplexes, ranged from 0.1 to 14.5 C, with thermal stability closely following the reassociation data. Reassociation experiments performed at 75 C, at which temperature only the more closely related DNA species form stable duplexes, gave similar indications of relatedness. At both temperatures, Alkalescens-Dispar strains showed close relatedness to E. coli, supporting the idea that they should be included in the genus Escherichia. Reciprocal binding experiments with E. coli BB, 02A, and K-12 yielded different reassociation values, suggesting that the genomes of these strains are of different size. The BB genome was calculated to be 9% larger than that of K-12, and that of 02A 9% larger than that of BB. Calculation of genome size for a series of E. coli strains yielded values ranging from 2.29 × 10⁹ to 2.97 × 10⁹ daltons. E. coli strains and closely related organisms were compared by Adansonian analysis for their relatedness to a hypothetical median strain. E. coli 0128a was the most closely related to this median organism. In general, these data compared well with the data from reassociation experiments among E. coli strains. However, anomalous results were obtained in the cases of Shigella flexneri, S. typhimurium, and “atypical” E. coli strains.     

Genome analysis of ground squirrels of the genus Citellus (Rodentia,Sciuridae) I. DNA reassociation kinetics and genome size of eight species

Kinetic of reassociation of short DNA fragments were measured in eight ground squirrel species: Citellus undulatus, C. parryi, C. relictus, C. dauricus, C. citellus, C. pygmaeus, C. fulvus and C. major. It was shown that 30–50% of their genome were represented by repeated sequences forming three kinetic fractions, i.e., very fast (Cot<10^-3), fast (Cot 10^-3–3×10^-1) and intermediate (Cot 6×10^-1–6×10¹). Based on parameters of DNA reassociation kinetics genome sizes of Citellus were estimated to range from 2.7 pg (C. dauricus) to 3.9 pg (C. pygmaeus and C. fulvus). Variation in genome sizes involves both the repeated and the non-repeated sequence components to approximately equal extents in all the species except C. dauricus. The linear quantitative relation between C-banding heterochromatin and both very fast and fast reassociated DNA fractions was established, but no connection with the intermediate fraction was found. No distinet relation was revealed between parameters of DNA reassociation kinetics and taxonomic status of species within genus or with the chromosome number of the karyotype.     

Differentiation of metaxylem cell line in the root ofAllium cepa

Summary The first step of differentiation in the root segments ofAllium cepa containing metaxylem cells in different stages of differentiation were studied by DNA reassociation curves and compared to meristem cell extracted DNA. Upon sonication of DNA samples to about 400 base pairs, the reassociation profiles of the heat denatured DNA, were spectrophotometrically followed at two different concentrations. The kinetic complexities,i.e., the number of base pairs per haploid genome of a given sequence and its redundancy were calculated. Differences were found at the level of highly and medium repetitive sequences, thus demonstrating that some DNA reassociation classes may undergo amplification during root development.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

Discrepancy in divergence of the mitochondrial and nuclear genomes ofDrosophila teissieri andDrosophila yakuba

Summary Restriction sites were compared in the mitochondrial DNA (mtDNA) molecules from representatives of two closely related species of fruit flies: nine strains ofDrosophila teissieri and eight strains ofDrosophila yakuba. Nucleotide diversities amongD. teissieri strains and amongD. yakuba strains were 0.07% and 0.03%, respectively, and the nucleotide distance between the species was 0.22%. Also determined was the nucleotide sequence of a 2305-nucleotide pari (ntp) segment of the mtDNA molecule ofD. teissieri that contains the noncoding adenine+thymine (A+T)-rich region (1091 ntp) as well as the genes for the mitochondrial small-subunit rRNA, tRNA^f-met, tRNA^gln, and tRNA^ile, and portions of the ND2 and tRNA^val genes. This sequence differs from the corresponding segment of theD. yakuba mtDNA by base substitutions at 0.1% and 0.8% of the positions in the coding and noncoding regions, respectively. The higher divergence due to base substitutions in the A+T-rich region is accompanied by a greater number of insertions/deletions than in the coding regions. From alignment of theD. teissieri A+T-rich sequence with those ofD. yakuba andDrosophila virilis, it appears that the 40% of this sequence that lies adjacent to the tRNA^ile gene has been highly conserved. Divergence between the entireD. teissieri andD. yakuba mtDNA molecules, estimated from the sequences, was 0.3%; this value is close to the value (0.22%) obtained from the restriction analysis, but 10 times lower than the value estimated from published DNA hybridization results. From consideration of the relationships of mitochondrial nucleotide distance and allozyme genetic distance found among seven species of theDrosophila melanogaster subgroup, the mitochondrial nucleotide distance observed forD. teissieri andD. yakuba is anomalously low in relation to the nuclear genetic distance.     

Deoxyribonucleic acid reassociation in the classification of flavobacteria.

DNA-DNA reassociation studies showed that Flavobacterium emningosepticum strains had a genetic relatedness of 91 to 100% with inter-strain duplexes having high thermal stabilities. The only exception, strain NCTC10016, had an average relatedness of only 43% to other strains of F. meningosepticum. The apparent divergence in DNA base sequence of this strain was reflected in the structural differences of some enzymes. There was a gradation of DNA relatedness among the Flavobacterium group II-b strains, but three strains were sufficiently related to constitute a species. Low levels of genetic relatedness were confirmed between F. meningosepticum and strains of Flavobacterium group II-b, group II-f, F. aquatile, F. breve, F. heparinum, F. pectinovorum, F. odoratum and Moraxella saccharolytica. All strains had base compositions in the range 32 to 46% guanine plus cytosine. The genome sizes of representative strains of F. meningosepticum and Flavobacterium group II-b were 2-50 X 10(9) to 3-52 X 10(9) daltons. The taxonomic implications of these findings are discussed.     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. IV. The F sequences in F14

The structure of F14, in particular the arrangement of the F sequences on this plasmid, has been studied by the electron microscope heteroduplex method. F14 has a molecular size of 311 ± 10 kilobase pairs (M = (206 ± 8) × 10⁶daltons). It contains all of F (94.5 kilobases). A sequence of length 5.7 kilobases, which occurs once in F (with co-ordinates 2.8 to 8.5F), is directly repeated in F14. It occurs at the two junctions of F DNA with chromosomal DNA. Thus, F14 contains about 211 ± 10 kilobases of chromosomal DNA. A previously unidentified direct repeat has also been discovered on F itself; the sequence with co-ordinates 93.2 to 94.5F is directly repeated at 13.7 to 15.0F. Physical observations indicate that the population of closed circular plasmid molecules extracted from F14-containing strains is heterogeneous. In addition to F14 itself, molecules the size of F and 2.3 times the size of F were found. The latter molecules contain all the chromosomal sequences of F14 and one copy of the 2.8 to 8.5F segment. Such heterogeneity was observed in both recA^? and recA⁺ backgrounds. It is proposed that this heterogeneity is due to intramolecular recombination events occurring within F14 between the duplicated 2.8 to 8.5F sequences. Such recombination can account for the previously observed genetic instability of F14. Another F prime plasmid, F186, independently isolated from the Hfr parent of AB313, was found to be identical to F14.     

DNA amounts in the nuclei ofParamecium aurelia andTetrahymena pyriformis

DNA amounts have been determined in the micronuclei and macronuclei of 8 strains ofParamecium aurelia and 6 strains ofTetrahymena pyriformis. In the case ofTetrahymena a distribution of values for the amount of DNA in the macronuclei of all the strains was observed but the lowest values were approximately the same, viz. 1.17×10⁻¹¹ g. There are two groups of strains in relation to micronuclear DNA values ofTetrahymena, one giving an average of 0.36×10⁻¹² g and the other 0.815×10⁻¹² g. The ratio of MIC/MAC DNA varies in the two groups.Paramecium again has a range of macronuclear values within each stock—lowest value 2.51×10⁻¹⁰ g—and the micronuclear values are similar in all stocks—approximately 0.613×10⁻¹² g. The ratio of MIC/MAC DNA is similar in each stock.—The haploid genome values calculated from these data show excellent agreement with the values obtained by DNA renaturation studies. Supported by a Research Grant B/SR/8276 from the Science Research Council. The Vickers densitometer was purchased with a grant from the Medical Research Council.     

Conserved structure of genes encoding components of botulinum neurotoxin complex M and the sequence of the gene coding for the nontoxic component in nonproteolyticClostridium botulinum type F

For investigation of the genes of proteins associated in vivo with botulinum neurotoxin (BoNT), polymerase chain reaction (PCR) experiments were carried out with oligonucleotide primers designed to regions of the nontoxic-nonhemagglutinin (NTNH) gene ofClostridium botulinum type C. The primers were used to amplify a DNA fragment from genomic DNA ofC. botulinum types A, B, E, F, G and toxigenic strains ofClostridium barati andClostridium butyricum. The amplified product from all of these strains hybridized with an internal oligonucleotide probe, whereas all nontoxigenic clostridia tested gave no PCR product and showed no reaction with the probe. TheNTNH gene was shown to be located upstream of the gene encoding BoNT, thereby revealing a conserved structure for genes encoding the proteins of the M complex of the progenitor botulinum toxin in these organisms. The sequence of theNTNH gene of nonproteolyticC. botulinum type F was determined by PCR amplification and sequencing of overlapping cloned fragments. NTNH/F showed 71% and 61% identity with NTNH ofC. botulinum type E and type C respectively.     

Deoxyribonucleic acid relatedness of some menaquinone-producing Flavobacterium and Cytophaga strains

Nine menaquinone-forming strains of the Flavobacterium-Cytophaga complex with DNA base compositions between 35 and 45 moles percent guanineplus-cytosine were investigated for genome sizes and DNA relatedness by DNA: DNA hybridization in vitro, using the optically recorded initial reassociation kinetics. Two strains representing C. hutchinsonii and C. marinoflava proved to be related on the 50 percent binding level, i.e. on a level of DNA relatedness commonly found within well-classified conventional genera of bacteria. Strains of C. johnsonae, F. heparinum, F. meningosepticum, F. odoratum, F. pectinovorum, and an unnamed Flavobacterium-Cytophaga strain were found to be interrelated, and linked to the genus Cytophaga, on the 30, or 20 percent binding levels, respectively. These findings indicate that the organisms in question are related to Cytophaga. They therefore should be transferred into the family Cytophagaceae.     

Plasmids controlling synthesis of hemolysin inEscherichia coli

Summary Plasmids of three different sizes, designated as plasmid A (mw: 65×10⁶), plasmid B (mw: 41×10⁶) and plasmid C (mw: 32×10⁶) respectively, have been isolated from various hemolytic wild-type strains ofE. coli. DNA-DNA hybridization was performed to determine their relationship. The wild-type strain, PM167a, harbours plasmids of all three sizes. Hybridization studies indicate that all three plasmids share extented sequence homologies but that plasmid A is not composed of plasmids B and C. Hybridization between plasmids of the donor strain and those of appropriate transconjugants demonstrates that in some cases plasmids with identical size are not longer completely homologous in their nucleotide sequences. This indicates that despite their defined sizes these plasmids are not stable genetic entities, but rather they undergo frequently recombination and dissociation during conjugation. In one particular transconjugant strain, K12-PM152/1, a plasmid D was found which is a stable recombined molecule of plasmids B and C of the original strain. Plasmids of size B found as the only extrachromosomal elements in a hemolytic wild-type strain (P224) and two transconjugant strains (e.g. K12-CM20 and K12-PM167/1) share extended nucleotide sequence homologies but are not identical. Little sequence homology was observed between two different hemolytic plasmids and the F and the Col Ib plasmids suggesting that the former do not belong to either the F-like or the I-like group of plasmids. Another hemolytic plasmid is F-like based on its sequence homologies with the F factor.     

Classification of the Legionnaires' disease bacterium: An interim report

Deoxyribonucleic acid (DNA) from strains of the Legionnaires' disease bacterium (LDB) was characterized in order to aid in the proper classification of this organism. The genome size of LDB DNA was estimated at 2.5×10⁹ daltons by reassociation kinetics; a guanine-plus-cytosine content of LDB of 39% was established by optical thermal denaturation and buoyant density ultracentrifugation measurements. DNA relatedness studies on 12 strains of the LDB indicated that they were all members of the same species. DNA relatedness studies have thus far failed to show that the LDB is significantly related to any other organism, including all members of Enterobacteriaceae,Pasteurella multocida, Francisella tularensis, Rochalimaea quintana, Vibrio species,Staphylococcus epidermidis, andFlavobacterium meningosepticum.     

Genetic relatedness of clinical and environmental isolates of lactose-positiveVibrio vulnificus

Environmental isolates of lactose-positiveVibrio vulnificus from different geographic areas were compared with clinical strains ofV. vulnificus on the basis of their phenotypic traits, virulence, DNA base composition, and DNA-DNA reasociation. EnvironmentalV. vulnificus strains were phenotypically indistinguishable from clinical isolates. These strains had a DNA base composition of 47–48 mol% guanine + cytosine and 85% reassociation at stringent temperature with DNA from clinicalV. vulnificus strains. These result indicateV. vulnificus strains from widely separated regions of the marine environment are indistinguishable from strains that have been agents of septicemia associated with shellfish consumption and of wound infections associated with seawater exposure.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

A simple linear transform for the Folin-Lowry protein calibration curve to 1.0 mg/ml

The stability of covalently mercurated DNAs during DNA:DNA reassociation, heteroduplex recovery on sulfhydryl-Sepharose, and S₁ nuclease digestion under a variety of solvent and temperature conditions is described. The nonspecific loss of²⁰³Hg from mercurated DNA can be minimized by use of aqueous formamide solvents in reassociation experiments and by minimizing exposure to sulfhydryl reagents and temperatures above 35°C. Single-stranded DNA is shown to be more sensitive to demercuration than is native, duplex DNA.     

Characterization of facultatively anaerobic marine bacteria belonging to group F of Lee,Donovan, and Furniss

Twenty-two isolates belonging to group F of Lee, Donovan, and Furniss were submitted to an extensive phenotypic characterization. The results of a numerical analysis of the data indicated that this group consisted of two biotypes which were phenotypically distinct from 22 previously characterized species ofBeneckea andPhotobacterium as well as from 3 species ofvibrio. The guanine-plus-cytosine content in the DNAs from 8 strains of group F was found to be 50.8±0.2 mol%. A set of 10 readily determinable phenotypic properties was proposed for the differentiation of strains of group F from other related species.