DNA-DNA Hybridization Studies Research Articles

Molecular cloning and characterization of chitinase genes from Streptomyces lividans 66

Summary: In order to study the genetic control of chitinolytic activity in Streptomyces, chitinase genes were cloned from S. lividans TK64 into the multicopy plasmid pIJ702 and their expression monitored in their natural host by measuring increases in chitinase productivity. Four independent clones were obtained, and the plasmids named pEMJ1, pEMJ5, pEMJ7 and pEMJ8. Restriction endonuclease digestion showed that although two of the plasmids (pEMJ7 and pEMJ8) shared a common DNA fragment, there were no substantial similarities between the inserts of plasmids pEMJ1, pEMJ5 and pEMJ7. This was confirmed by DNA-DNA hybridization studies. Four chitinases (A, B, C, and D) were identified, with molecular masses of 36, 46, 65, and 41 kDa, respectively. Production of chitinases A and B was specified by the plasmids pEMJ1 and pEMJ5, respectively. Genes for the other two chitinases (C and D) were carried by plasmid pEMJ7. Although significant differences were observed between chitinases A, B, and C in terms of optimum pH for activity and mode of digestion of substrates, chitinases C and D were very similar in these respects. Cloned genes were also expressed in S. coelicolor M130 and in Escherichia coli.

Genotypic heterogeneity of Flavobacterium group lib and Flavobacterium breve demonstrated by DNA‐DNA: hybridization

DNA-DNA hybridization studies on 42 stains presumptively identified as members of Flavobacterium group IIb and Flavobacterium breve indicated pronounced genotypic heterogeneity within these taxa. Three large groups highly related to the type strains of F. gleum, F. indologenes and F. breve respectively, and eight small groups were found. The group containing the type strain of F. breve was phenotypically indistinguishable from another genomic group, and these two groups were significantly separated from the other flavobacteria studied. The other nine genomic groups, representing Flavobacterium group IIb, could not with certainty be differentiated from each other by phenotypic characteristics, and there is no evidence indicating that these genomic groups differ from each other with respect to pathogenicity or ecology. Thus, it is suggested that for the time being the name "Flavobacterium group IIb" rather than specific epithets continue to be used for these bacteria.

Transfer of Brevibacterium divaricatum DSM 20297T, "Brevibacterium flavum" DSM 20411, "Brevibacterium lactofermentum" DSM 20412 and DSM 1412, and Corynebacterium lilium DSM 20137T to Corynebacterium glutamicum and Their Distinction by rRNA Gene Restriction Patterns

The results of DNA-DNA hybridization and chemotaxonomic studies indicated that the glutamic acid producers Brevibacterium divaricatum DSM 20297T (T=type strain), "Brevibacterium flavum" DSM 20411, "Brevibacterium lactofermentum" DSM 1412 and DSM 20412, Corynebacterium lilium DSM 20137T, and Corynebacterium glutamicum DSM 20300T and DSM 20163 are members of the same species. It is proposed that all of these strains should be classified in the species Corynebacterium glutamicum. Another glutamic acid-producing strain, Corynebacterium callunae DSM 20147T, was not related at the species level to C. glutamicum and should retain its separate species status. A restriction fragment length polymorphism analysis in which oligonucleotides targeted against conserved regions of 16S and 23S rRNA genes were used as hybridizing probes distinguished the individual strains. This method may be a helpful tool for strain identification.

Staphylococcus cohnii Subspecies: Staphylococcus cohnii subsp. cohnii subsp. nov. and Staphylococcus cohnii subsp. urealyticum subsp. nov.

Two major subspecies of Staphylococcus cohnii, namely S. cohnii subsp. cohnii, from humans, and S. cohnii subsp. urealyticum, from humans and other primates, are described on the basis of a study of 14 to 25 strains and 18 to 33 strains, respectively. DNA-DNA hybridization studies conducted in our laboratory in 1983 (W. E. Kloos and J. F. Wolfshohl, Curr. Microbiol. 8:115-121, 1983) demonstrated that strains representing the different subspecies were significantly divergent. S. cohnii subsp. urealyticum can be distinguished from S. cohnii subsp. cohnii on the basis of its greater colony size; pigmentation; positive urease, beta-glucuronidase, and beta-galactosidase activities; delayed alkaline phosphatase activity; ability to produce acid aerobically from alpha-lactose; and fatty acid profile. The type strain of S. cohnii subsp. cohnii is ATCC 29974, the designated type strain of S. cohnii Schleifer and Kloos 1975b, 55. The type strain of S. cohnii subsp. urealyticum is ATCC 49330.

Rhodoferax fermentans gen. nov., sp. nov., a phototrophic purple nonsulfur bacterium previously referred to as the ?Rhodocyclus gelatinosus-like? group

Strains of the phototrophic bacteria previously referred to as the “rhodocyclus gelatinosus-like (RGL)” group were taxonomically studied in comparison with Rhodocyclus species. Cells of the RGL strains were curved rods and motile by means of polar flagella. They contained bacteriochlorophyll a and carotenoids of the spheroidene series. The intracytoplasmic membrane system was absent. Photoorganotropho with various organic compounds as carbon sources was the preferred mode of growth. Aerobic growth at full atmospheric oxygen tension and fermentative growth under anaerobic-dark conditions were also possible. The major cellular fatty acids were palmitoleic acid and palmitic acid, and 3-hydroxylated fatty acids with octanoic acid predominating were also found. Both ubiquinone-8 and rhodoquinone-8 occurred as major quinones. The mol% guanine plus cytosine of the DNAs varied between 59.8 and 60.3. DNA-DNA hybridization studies showed that the RGL strains were highly related to each other but exhibited low levels of the homology to Rhodocyclus species. These data allow the establishment of the RGL group as a new taxon of the purple nonsulfur bacteria, for which the name Rhodoferax fermentans gen. nov., sp. nov. is proposed.

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.

Analysis of Actinobacillus pleuropneumoniae and Related Organisms by DNA-DNA Hybridization and Restriction Endonuclease Fingerprinting

The objective of this study was to determine the degree of genetic relatedness of Actinobacillus pleuropneumoniae to selected members of the family Pasteurellaceae, with particular emphasis on species commonly associated with swine. Free-solution DNA-DNA hybridization studies revealed that representative strains of all 12 serotypes of A. pleuropneumoniae formed a homogeneous group, sharing 74 to 90% sequence homology with A. pleuropneumoniae serotype 1. All serotypes of A. pleuropneumoniae tested demonstrated a high degree of genetic relatedness (66 to 79%) to the type species of the genus Actinobacillus, A. lignieresii. Little homology (less than 20%) was detected between A. pleuropneumoniae strains and selected Haemophilus spp. and Pasteurella spp. Since free-solution hybridization methods are technically demanding and require large amounts of highly purified DNA, restriction endonuclease fingerprinting (REF) was examined to determine whether it could be a useful taxonomic tool for classification of members of the family Pasteurellaceae. REF profiles were compared, and the degree of similarity between organisms was quantitated by calculating Jaccard similarity coefficients. There was a significant positive relationship between the REF Jaccard coefficients and the DNA homology values determined from free-solution hybridization experiments.

Comparative Study of Virion Structure, Protein Composition and Genomic DNA of Three Ascovirus Isolates

The virions of three ascoviruses isolated from the noctuids Heliothis zea, Spodoptera frugiperda and Trichoplusia ni were compared with respect to their size and structure, protein composition and the size and relatedness of their DNAs. The virions of the isolates from H. zea (HAV) and T. ni (TAV) were allantoid in shape (400 x 130 nm), enveloped and contained an inner particle which appeared to have an internal lipid bilayer surrounding the DNA core. The virions of the S. frugiperda isolate (SAV) were similar in structure and size, but were bacilliform in shape, and after formation, were often occluded in vesiculate occlusion bodies. In preparations of purified virions of each isolate, at least 12 polypeptides were detected that ranged in size from 10K to 200K and contained a major species of about 50K. The genome of SAV was about 140 kbp in size, whereas those of TAV and HAV were approximately 180 kbp. Analysis of DNA fragment patterns of the three isolates generated with BamHI, HindIII or XhoI, as well as DNA-DNA dot blot and Southern blot hybridization studies, demonstrated that HAV and TAV were closely related but not identical. The DNA from SAV, however, did not hybridize with the DNA from either of the other isolates. Thus the ascovirus isolates from T. ni and H. zea are considered variants of the same virus, whereas the isolate from S. frugiperda is a separate member of the ascovirus group.

Numerical Taxonomy of Mycobactin-Dependent Mycobacteria, Emended Description of Mycobacterium avium, and Description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov.

We performed a numerical taxonomy analysis of 38 Mycobacterium paratuberculosis and related mycobacterial strains, including wood pigeon mycobacteria; this analysis was based on 22 tests, which were selected for their potential discriminative value from a total of 51 tests studied and produced four well-defined clusters. Cluster 1 contained the M. paratuberculosis strains, including two strains isolated from Crohn's disease patients; cluster 2 contained Mycobacterium avium and Mycobacterium intracellulare reference strains; cluster 3 consisted of the wood pigeon mycobacteria; and the only strain in cluster 4 was M. paratuberculosis 316F, which is used for antigen and vaccine production. Strains in cluster 1 were mycobactin dependent even when they were subcultured, whereas strains in cluster 3 were unable to grow on egg medium and their growth was stimulated by pH 5.5. Growth stimulation by pyruvate, resistance to D-cycloserine (50 micrograms/ml), and alkaline phosphatase activity also were characteristics that were useful for discriminating between clusters 1 and 3. The results of previous DNA-DNA hybridization studies have demonstrated that M. avium Chester 1901, M. paratuberculosis Bergey et al. 1923, and the wood pigeon mycobacteria belong to a single genomic species, and we propose that the name of this species should be M. avium. On the basis of the results of previous genomic analyses based on restriction fragment length, the results of polymorphism studies, and DNA patterns determined by field inversion gel electrophoresis as well as the results of our phenotypic study, we propose that the species should be divided into subspecies which correspond to pathogenicity and host range characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Nesting Biology and Relationships of the Lesser Melampitta Melampitta lugubris

SummaryFrith, C. B. & Frith, D. W. (1990). Nesting biology and relationships of the Lesser Melampitta Melampitta lugubris. Emu 90, 65–73.Four active nests of the Lesser Melampitta Melampitta lugubris were studied in mixed lower montane forest at Tari Gap, Southern Highlands, Papua New Guinea. Nests, eggs, sexual dimorphism and parental care of this species are described for the first time. Iris colour is red in males, dark brown in females. Only females incubate and brood young but both sexes feed the nestling, male participation increasing with nestling age. The male regularly feeds the female close to and on the nest. Incubation period is at least 27–28 days and nestling period 35 days in one nest and at least 31 and 32 days in two others. DNA-DNA hybridisation studies suggest this species is a bird of paradise of the subfamily Paradisaeinae closest to Manucodia. Lesser Melampitta biology indicates, however, that it is unlike paradisaeine birds of paradise in most respects but is more like cnemophiline Cnemophilus macgregorii to which it would seem closest if its placement within Paradisaeidae is correct.

Extreme rates and heterogeneity in insect DNA evolution

DNA-DNA hybridization studies of insects, more specifically Drosophila and cave crickets, have revealed interesting patterns of genome evolution that contrast markedly with what has been seen in other taxa, especially mammals and birds. Insect genomes are composed of sections of single-copy DNA with extreme variation in rates of evolutionary change. This variation is more extreme than between introns and exons; introns fall into the relatively conserved fraction of the genome. Attempts to calculate absolute rates of change in Drosophila DNA have all led to estimates some 5-10 times faster than those found in most vertebrates; this is true even for the more conservative part of the nuclear genome. Finally we point out that morphological similarity, chromosomal similarity, and/or ability to form interspecific hybrids is often associated with quite high levels of single-copy DNA divergence in insects as compared to mammals and birds.

The future of DNA-DNA hybridization studies

This article draws on many vertebrate examples to assess the future of DNA-DNA hybridization studies. I first discuss whether applications of the method have reached the point of diminishing returns, or rather the start of a great leap forward, in our evolutionary understanding. Vertebrate groups whose relationships are especially likely to be illuminated include parrots, pigeons, bats, pinnipeds, mammalian carnivores, frogs, and rodents. There are at least two reasons why classifications based on DNA-DNA hybridization may prove to differ from classifications based on particular character, whether these be noncoding DNA sequences or protein sequences or anatomical characters. Because evolutionary relationships can now be deduced independently of anatomical characters, this should permit a renaissance in comparative anatomical studies of adaptation. The origin of major functional shifts from changes in a small fraction of the genome is illustrated by polar bears, sea otters, warblers, vultures, and especially by humans.

Lactoccus piscium sp. nov. a new Lactococcus species from salmonid fish

Molecular taxonomic studies were performed on ten strains of an unusual ‘viridans streptococcus’ that were originally isolated from human throats, blood and urine. On the basis of DNA-DNA hybridization studies the strains formed a single homology group distinct from all recognized species of oral and viridans streptococci. 16S ribosomal RNA reverse transcriptase sequence studies confirmed the genealogical distinctiveness of the human strains. The results of the present study clearly demonstrate that the human strains represent a new species of the viridans group for which the name Streptococcus parasanguis sp. nov. is proposed. The type strain is ATCC 15912.

Streptococcus parasanguissp. nov., an atypical viridansStreptococcusfrom human clinical specimens

Molecular taxonomic studies were performed on ten strains of an unusual 'viridans streptococcus' that were originally isolated from human throats, blood and urine. On the basis of DNA-DNA hybridization studies the strains formed a single homology group distinct from all recognized species of oral and viridans streptococci. 16S ribosomal RNA reverse transcriptase sequence studies confirmed the genealogical distinctiveness of the human strains. The results of the present study clearly demonstrate that the human strains represent a new species of the viridans group for which the name Streptococcus parasanguis sp. nov. is proposed. The type strain is ATCC 15912.

Taxonomic Study of Anaerobic, Gram-Negative, Rod-Shaped Bacteria from Breweries: Emended Description of Pectinatus cerevisiiphilus and Description of Pectinatus frisingensis sp. nov., Selenomonas lacticifex sp. nov., Zymophilus raffinosivorans gen. nov., sp. nov., and Zymophilus paucivorans sp. nov.

A collection of 47 strains of obligately anaerobic, gram-negative, rod-shaped bacteria that were isolated mainly from spoiled beer and pitching yeast was studied to learn more about their taxonomic positions. A new species of the genus Pectinatus, Pectinatus frisingensis, a new species of the genus Selenomonas, Selenomonas lacticifex, and a new genus comprising two species, Zymophilus raffinosivorans and Zymophilus paucivorans, are described. All of the strains contained directly cross-linked meso-diaminopimelic acid-containing peptidoglycan and in addition the diamine cadaverine or (rarely) putrescine. The diamine was covalently linked to the alpha-carboxyl group of D-glutamic acid in the peptide subunit of peptidoglycan. Lipid F was also found as a characteristic cellular compound. The phylogenetic relationships of members of these new species were examined by reverse transcriptase sequencing of 16S rRNA or by DNA-DNA hybridization studies or both. All of the organisms belong to the subdivision containing species with gram-negative cell walls within the phylum of gram-positive bacteria. This finding is in good agreement with the presence of a peptidoglycan that contains diamine.

Taxonomy of Alkaliphilic Bacillus Strains

The DNA base compositions of 78 alkaliphilic Bacillus strains were determined. These strains were grouped as follows: DNA group A, guanine-plus-cytosine (G+C) content of 34.0 to 37.5 mol% (17 strains); DNA group B, G+C content of 38.2 to 40.8 mol% (33 strains); and DNA group C, G+C content of 42.1 to 43.9 mol% (28 strains). DNA group A includes the type strain of Bacillus alcalophilus Vedder 1934. DNA-DNA hybridization studies with DNA group A strains revealed that only one strain, strain DSM 2526, exhibited a high level of DNA homology with B. alcalophilus DSM 485T (T = type strain). Neither strain DSM 485T nor any other DNA group A strain is homologous to any of the Bacillus type strains with comparable base compositions. Six strains formed a distinct group containing three highly homologous strains and three strains exhibiting greater than 50% DNA homology.

Isolation and Characterization of Methanobacterium espanolae sp. nov., a Mesophilic, Moderately Acidiphilic Methanogen

Bacterial strain GP9T (T = type strain), a nonmotile, nonsporeforming, mesophilic, methanogenic bacterium, was isolated from the primary sludge obtained from the waste treatment facility of a major kraft pulp mill in Canada. Single cells were 6.0 by 0.8 μm and stained gram positive. Growth and methane production occurred only with H2-CO2 as the substrate. Acetate, formate, propionate, butyrate, pyruvate, methanol, or trimethylamine could not serve as a sole source of carbon and energy for growth. The optimum pH for growth was between 5.6 and 6.2; consistent growth and methane production were not observed below pH 4.68. The optimum temperature for growth was 35°C, and little or no growth was observed during incubation at 15 and 50°C. Kanamycin and bacitracin were severe inhibitors of growth and methanogenesis, whereas 100 μM bromoethanesulfonic acid caused 30% inhibition. Supernatant from primary sludge enhanced growth by about 10%. The DNA base composition was 34 mol% guanine plus cytosine. On the basis of physiological characteristics, indirect immunofluorescence typing, and DNA-DNA hybridization studies, the isolate is named Methanobacterium espanolae sp. nov.

Enzymatic profiles of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillae

The enzymatic activities of 39 strains of Erysipelothrix rhusiopathiae and 34 of E tonsillae were determined with the API ZYM system. The profiles of these two species were very similar, differing solely in N-acetyl-beta-glucosaminidase activity. Whereas 90 per cent of strains of E rhusiopathiae exhibited strong activity with N-acetyl-beta-glucosaminidase, positive reactions were observed for this enzyme in only 24 per cent of strains of E tonsillae. These results support previous DNA-DNA hybridisation studies and suggest that E tonsillae is a new species of the genus Erysipelothrix.

Random insertion of the gentamicin resistance transposon Tn4001 in Mycoplasma pulmonis

The staphylococcal transposon Tn4001 was introduced into Mycoplasma pulmonis using an Escherichia coli-derived vector by polyethylene glycol-mediated transformation. Using a reaction mixture containing 10 micrograms plasmid DNA, 10 micrograms yeast tRNA, and 34-35% polyethylene glycol per 1 x 10(8) cells, Tn4001 could be introduced into M. pulmonis at a frequency of 5 x 10(-5) per colony forming unit. DNA-DNA hybridization studies illustrated that Tn4001 could occupy a diversity of insertion sites in the M. pulmonis chromosome. These data indicated that Tn4001 is a potentially useful tool for the introduction of mutations and for genetic studies in M. pulmonis.

Streptococcus vestibularis sp. nov. from the Human Oral Cavity

Thirteen strains of gram-positive, catalase-negative, chain-forming cocci isolated from the vestibular mucosa of human oral cavities were compared with other oral streptococcal species. The new strains were unusual in that they formed acid from lactose, from salicin, and usually from cellobiose, but not from mannitol, sorbitol, inulin, or raffinose, and infrequently formed acid from trehalose. They hydrolyzed esculin, urea, and starch but not arginine, formed hydrogen peroxide, and were usually Voges-Proskauer positive. They did not produce extracellular glucan or fructan from sucrose. The strains were compared by analyzing long-chain fatty acids, by determining whole-cell polypeptide patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by performing deoxyribonucleic acid (DNA)-DNA hybridizations. They possessed fatty acid profiles with major amounts of hexadecanoic (C16:0) and octadecenoic (C18:1ω7; cw-vaccenic) acids together with tetradecanoic (C14:0; myristic), hexadecenoic (C16:1; palmitoleic), octadecanoic (C18:0; stearic), octadecenoic (C18:1ω9; oleic), and eicosenoic (C20:1) acids, as shown by capillary gas-liquid chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis polypeptide patterns of the isolates showed some similarity to the patterns obtained for strains of Streptococcus salivarius subsp. salivarius, although they were, nevertheless, clearly distinguishable from the latter. DNA-DNA hybridization studies demonstrated that the new strains are more closely related to S. salivarius than to the other species of oral streptococci, but are sufficiently dissimilar to warrant separate species status. The name Streptococcus vestibularis is proposed. The DNA base composition is 38 to 41 mol% guanine plus cytosine. The type strain is strain MM1 (= NCTC 12166).