Chromosomes,DNA sequences,and evolution in salamanders of the genus Plethodon

Chromosomes and DNA sequence homologies have been studied in 15 species of North American salamander belonging to the genus Plethodon. These include 4 Eastern small species, 5 Eastern large species, 5 Western, and 1 New Mexican species. All species have 14 metacentric or sub-metacentric chromosomes. Their karyotypes are closely similar, but their C values range from 18–69 pg. DNA:DNA molecular hybridization studies showed that salamanders belonging to the same species group had between 60 and 90% of the observed repetitive DNA sequences in common, different groups of Eastern species had between 40 and 60% in common, and Eastern and Western groups had less than 10% in common. The slowly reassociating DNA sequences were also diverse among species, but higher levels of homology were observed than in the case of repetitive sequences. The New Mexican species was exceptional in showing little homology with other species with respect to either repetitive or slowly reassociating sequences.     

Anatomy of mitochondrial DNA from Paramecium aurelia

Summary The linear genome of mitochondrial DNA from four species of Paramecium aurelia was investigated with respect to restriction endonuclease fragments, location and number of ribosomal RNA genes, and interspecies EcoRI and HindIII fragment homologies. One copy of each of the rRNA genes was found in all four species and the 14s and 20s rRNA genes were separated by at least 3,000 bp. R-Loop analysis of the 20s rRNA gene did not reveal the presence of an intervening sequence. Interspecies homology studies showed species 1, 5, and 7 to have a high degree of homology but species 4 was less than 50% homologous to species 1 mt DNA. For all four species, rRNA genes showed good homology indicating that these DNA sequences are highly conserved, even between species having many non-homologous regions. A major region of DNA which displayed little homology between species 1 and 4 was that fragment containing sequences essential for initiation of DNA replication.     

Molecular Heterogeneity of the Bdellovibrios: Evidence of Two New Species

A systematic examination of a variety of isolates of the bacterial endoparasite Bdellovibrio has revealed extensive molecular diversity. The quantity of deoxyribonucleic acid (DNA) polynucleotide homology ranges from more than 90% among the isolates with DNA containing 50 to 51% guanine plus cytosine (GC) to undetectable levels between the 43% GC and 51% GC isolates. The two isolates with low GC-containing DNA (H-I Bdellovibrio A3.12 and UKi2) have only 16% DNA homology. H-I Bdellovibrio A3.12 and 109 have barely detectable ribosomal ribonucleic acid (rRNA) homology, whereas the homology approaches 100% among all the high GC isolates tested. Cases of high DNA/DNA and DNA/rRNA homologies are reflected in low dissimilarities of enzyme migration patterns in starch gel electrophoresis. The dissimilarities exhibited among the high GC Bdellovibrio isolates are as low as those previously reported for different Escherichia coli strains. The zymograms of H-I Bdellovibrio A3.12 and UKi2 are completely different from each other as well as from all other bdellovibrios (100% dissimilarity). Genome sizes determined for the representative isolates demonstrate three size ranges which coincide with group differences based on the above measurements. Enzyme assays reveal that all isolates possess a tricarboxylic acid cycle and most contain an alanine and glutamic dehydrogenase. We conclude that the use of bacterial endoparasitism as a defining trait has resulted in a molecularly diverse collection of isolates. It is recommended that the specific epitaph bacteriovorus be used only for the type specimen (Bdellovibrio 100 of Stolp and Starr, 1963) and for other related 50 to 51% GC isolates. The heterogeneity of the group warrants two new species. We designate Bdellovibrio A3.12 as the nomenclatural type of B. starrii sp. n. and Bdellovibrio UKi2 as the nomenclatural type of B. stolpii sp. n.     

DNA base sequence homologies among strains of Streptococcus sanguis.

DNA was isolated from 19 strains and substrains of Streptococcus sanguis and analysed for guanine plus cytosine (GC) contents and base sequence homologies. Three groups could be discerned: group 1 strains had 40-8 to 42-8 mol % GC; group 2, 42-7 to 44-0 mol % GC; group 3, 43-8 to 46-4 mol % GC. DNA homologies between groups 1 and 3 were 40 to 60% at 67 degrees C and 40% at 72 degrees C. The homologies of group 2 towards groups 1 and 3 were much lower. Strains in groups 1 and 3 hydrolysed arginine and aesculin and fermented inulin; group 2 strains did not. Groups 1 and 3 could be considered subspecies of S. sanguis. Group 2 should not be considered S. sanguis.     

Streptococcus uberis: an Approach to its Classification

Thirty-one strains of streptococci, selected because they were considered to be or were similar to Streptococcus uberis , were examined by physiological tests and for properties of their lactate dehydrogenases (LDHs) and deoxyribonucleic acid (DNA). Twenty-nine of the 31 strains fell into two main genotypes as judged by DNA/DNA hybridization. One genotype (Group I) included the type strain of Strep. uberis and most other strains in this group were physiologically similar to it and had the same percent guanine + cytosine (%GC) in their DNA. The other genotype (Group II) contained strains which were more variable physiologically and had DNA of a lower % GC than the type strain. The European strains examined contained two distinct LDH types, one being associated with the Group I and the other with Group II strains. Three American strains examined however had LDH's which were unlike those of the European strains.     

Clostridium sporogenes isolates and their relationship to C. botulinum based on deoxyribonucleic acid reassociation.

Sixty-two isolates of Clostridium sporogenes from canned foods were examined for cultural properties, heat resistance and DNA-DNA homology to Clostridium botulinum type A190. Sporulation was observed in most of 21 umbonate and rhizoidal colony-forming strains (colony-type I strains), but not in most of the 41 strains with convex and circular or crenate colonies with a mat to semi-glossy surface (colony-type II strains). More than half of the latter strains showed much higher heat resistance than the rhizoidal colony-forming strains. The DNA isolated from colony-type II strains was 81% or more homologous to C. botulinum A190 DNA, forming duplexes which had thermostabilities similar to homologous duplexes of strain A190 DNA. Colony-type I strains differed from C. botulinum by 30 to 40% DNA homology and the DNA duplexes formed between these strains and strain A190 showed deltaT m(e) values of 7-0 degrees C when compared with the T m(e) of homologous DNA duplexes of strain A190.     

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA-DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA-DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid.     

Characterization of Deoxyribonucleic Acids from Streptomycetes and Nocardiae

The relationships among selected streptomycetes, nocardiae, and mycobacteria have been determined, based upon the base composition of their deoxyribonucleic acid (DNA) and upon the ability of their denatured DNA to anneal with single-stranded reference DNA. The streptomycetes constituted a homogeneous group whose DNA contained between 69 and 73 mole% guanine + cytosine (% GC). Moreover, the streptomycetes examined showed 37 to 88% homology with the Streptomyces venezuelae and S. rimosus reference DNA. The nocardial and mycobacterial DNA both contained 62 to 69% GC. The nocardial strains studied fell into either a 62 to 64% GC group or a 68 to 69% GC group, indicating that they should not be assigned to a single species. The nocardiae having 68 to 69% GC showed 24 to 44% homology with S. venezuelae reference DNA. In competition experiments, wherein unlabeled heterologous DNA interfered with binding of labeled homologous DNA, the nocardial DNA with 68 to 69% GC showed a greater degree of homology with the streptomycetes than did the nocardial DNA with 62 to 64% GC. In addition, the DNA from spores of S. venezuelae was cursorily examined, and interactions between S. venezuelae denatured DNA and polyribonucleotides were sought. The buoyant density of the DNA from S. venezuelae spores was distinctly less than that from mycelia. Moreover, denatured S. venezuelae DNA formed a dense complex with polyriboguanylate.     

Reclassification of Pichia membranifaciens sensu Kurtzman

Forty strains which were stocked as Pichia membranifaciens sensu Kurtzman and 9 strains stocked as Candida valida, anamorphs of P. membranifaciens, in the Institute for Fermentation, Osaka (IFO) were reclassified based on the data of base composition of nuclear DNA, DNA/DNA hybridization, coenzyme Q system, electrophoretic karyotype, and base sequence of 18S rDNA. P. membranifaciens complex was assigned into 3 groups: (I) P. membranifaciens group, including 25 strains with high DNA homologies to the type strain of P. membranifaciens (72–98%); (II) P. manshurica group, including 18 strains with high DNA homology of 79–95% to the type strain of P. manshurica; and a group including the remaining 6 strains, which had low DNA homology to the above two species. GC content was 42.9–45.3 mol% for the P. membranifaciens group, 40.0–42.0 mol% for the P. manshurica group, and 27.2–44.7 mol% for the remaining group. All three groups had ubiquinone Co Q-7. Of the 6 anomalous strains, IFO 0162 was identified as Pichia deserticola, and IFO 0839 and IFO 0840 were identified as Issatchenkia occidentalis; but IFO 0842, IFO 0843, and IFO 1788 were thought to be unknown strains.     

Taxonomic relationships among Clostridium novyi Types A and B, Clostridium haemolyticum and Clostridium botulinum type C

The present study was undertaken to examine the genetic relationships among the closely related species, Clostridium novyi types A and B, C. haemolyticum and C. botulinum type C. These species were tested for DNA-DNA homology and thermostability of DNA duplexes and sorted into three genetically related groups: I, C. novyi type A; II, C. novyi type B, C. haemolyticum and one C. botulinum type C strain (Stockholm); III, the remaining C. botulinum type C strains. A few biochemical criteria corresponding to the genetic differences were recommended to differentiate each group. These studies imply that C. haemolyticum might be considered as C. novyi type D and that there are two genetically different groups in C. botulinum type C.     

Molecular relationship between two groups of the genus Leptospirillum and the finding that Leptospirillum ferriphilum sp. nov. dominates South African commercial biooxidation tanks that operate at 40 degrees C

Iron-oxidizing bacteria belonging to the genus Leptospirillum are of great importance in continuous-flow commercial biooxidation reactors, used for extracting metals from minerals, that operate at 40 degrees C or less. They also form part of the microbial community responsible for the generation of acid mine drainage. More than 16 isolates of leptospirilla were included in this study, and they were clearly divisible into two major groups. Group I leptospirilla had G+C moles percent ratios within the range 49 to 52% and had three copies of rrn genes, and based on 16S rRNA sequence data, these isolates clustered together with the Leptospirillum ferrooxidans type strain (DSM2705 or L15). Group II leptospirilla had G+C moles percent ratios of 55 to 58% and had two copies of rrn genes, and based on 16S rRNA sequence data, they form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present among the leptospirilla tested, with two DNA-DNA hybridization similarity subgroups found within group I. The two groups could also be distinguished based on the sizes of their 16S-23S rRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a separate species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and by carrying out restriction enzyme digests of the products. Several, but not all, isolates of the group II leptospirilla, but none from group I (L. ferrooxidans), were capable of growth at 45 degrees C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.     

DNA homology study of Corynebacterium diphtheriae v. gravis groups I, II and III, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (ovis)

The homology of genomes within Krylova 's groups I, II and III of C. diphtheriae, including toxigenic C. diphtheriae and their nontoxigenic precursors within the same group, was confirmed by the method of DNA/DNA molecular hybridization; the homology of DNA within the groups was 89-103%, the thermostability of heteroduplexes being high (on the level of homoduplexes ). The heterogeneity of genomes within these 3 groups of cultivar gravis was confirmed, which made it possible to consider C. diphtheriae, groups I, II and III, to belong to different, though closely related species; in intergroup hybridization the homology of DNA varied, as a rule, between 66% and 73%, while the thermostability of heteroduplexes was low: delta T50 was -3 degrees C to -6 degrees C. The differences in genomes (on the level of different species) between 3 groups of C. diptheriae v. gravis on one hand and C. diphtheriae v. mitis C7 (-) tox- and its convertant C7 (beta) tox+ of phage tox+ on the other hand (DNA homology being 56-62%), as well as between C. diphtheriae v. intermedius No. 328 tox+ on one hand and the representatives of 3 groups of C. diphtheriae v. gravis and C. diphtheriae v. mitis, strain C7 (beta) tox+, on the other hand (DNA homology being 42-43%) were revealed. The heterogeneity of genomes (on the level of different genera) was revealed between C. diphtheriae strains, cultivars gravis (groups I, II and III), mitis (C7(-) tox- and C7 (beta) tox+) and intermedius (No. 328 tox+) on one hand and C. ulcerans and C. pseudotuberculosis (ovis) strains on the other hand; DNA homology was 11-17% for C. ulcerans and 22-26% for C. pseudotuberculosis (ovis), the thermostability of heteroduplexes being at the lowest level (delta T50 was -11 degrees C to -13 degrees C). As a result, C. diphtheriae, classified by Bergey as a single species, was found to comprise 5 species detected by means of marking in accordance with their phenotypical features and genome structure, carried out by the method of DNA/DNA molecular hybridization; among these species were group I, II and III strains of cultivar gravis, strain C7 of cultivar mitis and strain No. 328 of cultivar intermedius. C. ulcerans and C. pseudotuberculosis (ovis) strains investigated in this study can possibly be placed outside the genus including 5 C. diphtheriae species.     

Clostridia from Grana Cheese

Forty strains of proteolytic and saccharolytic clostridia isolated from Grana cheese were re-identified by DNA-DNA homology. Thirty culture collection strains were also examined for comparison. Organisms of the species Clostridium tyrobutyricum which present variability in phenotypic characteristics were found to constitute a homogeneous group, genetically unrelated to all the reference or competitor strains used. The proteolytic clostridia from cheese show a high level of homology with the reference strains C.sporogenes ATCC 319 and C. sporogenes ATCC 3584 and negligible homology with C. bifermentans ATCC 19299. Three strains with phenotypic characters very similar to those of the species C. butyricum present a level of DNA homology with C. butyricum ATCC 19398 ranging from 61–71%. In the light of the results obtained some taxonomic conclusions have been drawn.     

Genetic diversity ofRhizobium strains which nodulateLeucaena leucocephala

DNA homologies at 65°C in 0.14 phosphate buffer, pH 6.8, were determined between 24 strains ofRhizobium capable of nodulatingLeucaena leucocephala and fourRhizobium reference strains. Twenty-one strains (88%) were placed in one of four DNA homology groups. The mean relative homology within a group was 65%, while the mean relative homology between groups was 20%. Thermal melting points for reassociated DNA (ΔT_m(e) values) were also measured. The lack of DNA homology between groups indicates that several very different populations of bacteria are capable of nodulating and fixing nitrogen with leucaena.     

Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.     

Development and validation of PCR primers to assess the diversity of Clostridium spp. in cheese by temporal temperature gradient gel electrophoresis

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.     

Molecular Relationship between Two Groups of the Genus Leptospirillum and the Finding that Leptospirillum ferriphilum sp. nov. Dominates South African Commercial Biooxidation Tanks That Operate at 40°C

Iron-oxidizing bacteria belonging to the genus Leptospirillum are of great importance in continuous-flow commercial biooxidation reactors, used for extracting metals from minerals, that operate at 40°C or less. They also form part of the microbial community responsible for the generation of acid mine drainage. More than 16 isolates of leptospirilla were included in this study, and they were clearly divisible into two major groups. Group I leptospirilla had G+C moles percent ratios within the range 49 to 52% and had three copies of rrn genes, and based on 16S rRNA sequence data, these isolates clustered together with the Leptospirillum ferrooxidans type strain (DSM2705 or L15). Group II leptospirilla had G+C moles percent ratios of 55 to 58% and had two copies of rrn genes, and based on 16S rRNA sequence data, they form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present among the leptospirilla tested, with two DNA-DNA hybridization similarity subgroups found within group I. The two groups could also be distinguished based on the sizes of their 16S-23S rRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a separate species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and by carrying out restriction enzyme digests of the products. Several, but not all, isolates of the group II leptospirilla, but none from group I (L. ferrooxidans), were capable of growth at 45°C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.     

A thermal denaturation study of the genomic DNAs from the North American genera of the fish family Percidae

The genomic DNAs of 1 1 species of percid fishes representing the five recognized North American genera are characterized using data from thermal denaturation assays. Base compositions were estimated from the transitional melting temperature of native and sonicated DNA and expressed as per cent guanine-cytosine (%GC) values. Among genera, %GC values for native DNAs (c, 23,000 base pairs in length) range between 38.3% GC for yellow perch, Perca flavescens (Mitchill), to 43.2% GC for sauger, Stizostedion cunadense (Smith). Significant variation in %GC values was observed among surveyed genera of the subfamily Percinae, which include Perca, Percinu, Etheostoma and Ammocrypfa . Melting profiles were generated for each species, and distinct GC rich regions were identified within the genomes of walleye, Sfizostcdion virreum (Mitchill) and Etheostoma spp. Compositional heterogeneity (CH) and asymmetry values were calculated from melting profile data. Patterns of variation in genomic characters differed among the genera surveyed. Members of the speciose genus Etheostomu showed relatively little variation in genomic characters, whereas Stizosredion exhibited significant interspecific variation.     

A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov.

Sixty-one strains of the root-associated nitrogen fixer Spirillum lipoferum exhibited a similar morphology in peptone--succinate salts medium: vibrioid cells having a diameter of 1.0 micrometer. When grown in broth the cells had a single polar flagellum, but when grown on agar at 30 degrees C lateral flagella of shorter wavelength were also formed. The DNA base composition was 69--71 mol% guanine + cytosine when determined by thermal denaturation. DNA homology experiments indicated the occurrence of two distinct but related homology groups: 46 strains were in group I and 15 strains were in group II. Group II strains were distinguished by their ability to use glucose as a sole carbon source for growth in nitrogen-free medium, by their production of an acidic reaction in a peptone-based glucose medium, by their requirement for biotin, and by their formation of wider, longer, S-shaped or helical cells in semisolid nitrogen-free malate medium. The results indicate that two species exist, and on the basis of their characteristics it is proposed that they be assigned to a new genus, Azospirillum. Strians belonging to group II are named A. lipoferum (Beijerinck) comb. nov., while those belonging to group I are named A. brasilense sp. nov. Strain Sp 59b (ATCC29707) is proposed as the neotype strain for A. lipoferum, and strain Sp 7 (ATCC 29145) is proposed as the type strain for A. brasilense.     

Deoxyribonucleic Acid Base Composition and Homology Studies of Leptospira

Four distinct genetic groups of leptospiras were demonstrated among selected pathogenic and "biflexa" serological types. Pathogenic leptospiras could be divided into two groups on the basis of per cent guanine + cytosine (GC) in their deoxyribonucleic acid (DNA). One group had 36 +/- 1%, the other 39 +/- 1%. The biflexa strains had DNA of 39 +/- 1% GC, but were further separated into two groups on the basis of DNA-annealing tests. Strains within groups had a high degree of specific duplex formation (75% binding or more with reference to the homologous DNA). There was little or no genetic relatedness between strains of the four groups (less than 10% DNA homology). The thermal elution midpoint of heterologous DNA duplexes was always lower than the homologous reaction. The serological relationships among strains were not meaningful in terms of relatedness determined by specific duplex formation.