rDNA Restriction Patterns Research Articles

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber Apostichopus japonicus

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes (protease, amylase, and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus, which can produce protease, amylase, and cellulase. Virgibacillus, however, can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.

Biocontrol potential and polyphasic characterization of novel native Trichoderma strains against Macrophomina phaseolina isolated from sorghum and common bean

Native strains of Trichoderma isolated from sorghum and common bean crop soils were investigated to assess their biocontrol potential over the phytopathogenic fungus Macrophomina phaseolina, isolated from diseased plants. The Trichoderma strains were characterized with a polyphasic approach, which combined the analysis of their morphological characteristics, enzymatic activity, macro- and microculture test results, rDNA restriction patterns (AFLP), ITS1-5.8S-ITS2 rDNA sequences, and protein profiles. The integration of these data sets can be used to select new isolates as biological control agents against native fungal phytopathogens. In general, we observed a positive correlation between the secretion of beta-1,3-glucanase and N-acetylhexosaminidase, and the biocontrol capacities of all the Trichoderma isolates. Strains with the best hyperparasitic behavior against M. phaseolina isolated from diseased bean and sorghum were Trichoderma sp. (TCBG-2) and Trichoderma koningiopsis (TCBG-8), respectively.

Bacterial Genus Xenorhabdus (Enterobacteriaceae) Part of the Bacterial Complex of Five Bulgarian Populations Entomopathogenic Nematode from Genus Steinernema (Steinernematidae: Rhabditida)

ABSTRACTTwenty one bacterial strains of Xenorhabdus obtained from five nematode hosts from genus Steinernema were typed by analyzing 16S rDNA restriction patterns obtained after digestion of PCR amplified 16S rDNAs. Three tetramer endonucleases were used. A total of 14 genotypes were identified, forming main clusters after analysis using arithmetical methods (UPMGA). To identify the strains and all genotypes, the enzymes Hae III, Alu I and Hha I were used in the analysis. From our results we could conclude that RFLP-PCR of 16S rDNA analysis is an accurate method for identifying enomopathogenic nematode bacterial symbionts.

Isolation and characterization of bacteria from the rhizosphere of wheat

In this study, we isolated bacteria from rhizosphere and endorhizophere of wheat crops of the central region of Argentina. The isolates were phenotypically characterized and the restriction patterns of 16S rDNA (ARDRA) using endonuclease AluI were analysed. Representative isolates were used to evaluate the effect of the inoculation on the growth of wheat under greenhouse conditions. The effects of plant growth-promoting bacteria on wheat plants were studied by evaluating shoot fresh and dry weights and root fresh and dry weights. One native strain increased the shoot and root dry biomass by 23% and 45% respectively. Other strains increased the shoot dry biomass. A 1.5 kb fragment of the 16S rRNA gene of one isolate was sequenced. This isolate showed high identity with different species of Pseudomonas.

Chromosomes and genetic organization of rdna in planarians of the genus schmidtea (platyhelminthes, tricladida)

The genus Schmidtea includes four planarian species characterized by karyological differentiation. Different levels of ploidy also define various biotypes, typically associated with sexual or pseudogamic forms of reproduction. The present paper gives a karyological survey of somatic cells and unfertilized oocytes of specimens collected from natural populations. With the aim of integrating classical karyological analyses with molecular data, the molecular structure of ribosomal DNA (rDNA), in individuals of different species and biotypes is also compared, using restriction enzyme analysis. The results reveal the presence of species‐specific rDNA restriction patterns. In spite of the occurrence of poly‐ploid forms, a high level of structural intraspecific homogeneity of these genes was also observed among different populations.

Concerted evolution of 18-5.8-26S rDNA repeats in Nicotiana allotetraploids

We review and extend data showing concerted evolution of parental 18-5.8-26S nuclear ribosomal DNA (18-26S rDNA) gene families in three natural Nicotiana allotetraploids (N. tabacum, N. rustica and N. arentsii, each 2n = 4x = 48) and one synthetic N. tabacum line (Th37, ♀ N. sylvestris (2n = 24) x ♂ N. tomentosiformis (2n = 24)). The origin of the gene families was analysed by sequence polymorphisms in the intergenic spacer (IGS) region and the number of chromosomal loci by fluorescence in situ hybridization (FISH). FISH revealed that the number and locations of 18-26S rDNA in the natural allopolyploids was the sum of those found in the diploid progenitors. However, the rDNA restriction patterns showed polymorphisms in the IGS that were not additive, suggesting that parental rDNA clusters were partially (N. tabacum, N rustica) or completely (N. arentsii) overwritten by hybrid-specific units. Thus the Nicotiana allotetraploids show evidence of concerted evolution, including both intralocus and interlocus gene conversion. A feral N. tabacum collected in Bolivia had a higher proportion of unconverted parental rDNA units than cultivated tobacco varieties, suggesting either that rDNA homogenization is accelerated by inbreeding or multiple origins of tobacco. There is no evidence for the elimination of N. sylvestris-derived rDNA units in the synthetic Th37 tobacco line as occurred in natural tobacco, although several novel rDNA unit variants were found in most but not all the hybrid plants. Factors that may control the occurrence and extent of rDNA homogenization are discussed for allopolyploids in Nicotiana and other taxa.

Burkholderia unamae sp. nov., an N2-fixing rhizospheric and endophytic species.

It was shown recently that the genus Burkholderia is rich in N2-fixing bacteria that are associated with plants. A group of these diazotrophic isolates with identical or very similar 16S rDNA restriction patterns [designated amplified rDNA restriction analysis (ARDRA) genotypes 13, 14 and 15] was selected and a polyphasic taxonomic study was performed, which included new isolates that were recovered from rhizospheres, rhizoplanes or internal tissues of maize, sugarcane and coffee plants. Morphological, physiological and biochemical features, as well as multi-locus enzyme electrophoresis profiles and whole-cell protein patterns, of 20 strains were analysed. In addition, analysis of cellular fatty acid profiles, 16S rDNA sequence analysis and DNA-DNA reassociation experiments were performed with representative strains. The taxonomic data indicated that the strains analysed belong to a novel diazotrophic Burkholderia species, for which the name Burkholderia unamae sp. nov. is proposed. Strain MTl-641T (=ATCC BAA-744T=CIP 107921T), isolated from the rhizosphere of maize, was designated as the type strain. B. unamae was found as an endophyte of plants grown in regions with climates ranging from semi-hot subhumid to hot humid, but not from plants grown in regions with semi-hot or hot dry climates. Moreover, B. unamae was isolated from rhizospheres and plants growing in soils with pH values in the range 4.5-7.1, but not from soils with pH values higher than 7.5.

Identification of extracellular enzyme producing alkalophilic bacilli from Izmir province by 16S-ITS rDNA RFLP.

To screen industrially important extracellular enzymes from the newly isolated alkalophilic bacilli and to characterize them by phenotypic and 16S-internal transcribed spacer (ITS) rDNA restriction pattern analysis. Three different environmental samples, soil, leather and horse faeces, were collected within the province of Izmir. Isolates grown on Horikoshi-I medium for 24 h at 37 degrees C were screened for extracellular enzyme activity by using eight different substrates: birchwood xylan, carboxymethylcellulose, casein, citrus pectin, polygalacturonic acid, soluble starch, and Tween 20 and 80. In total, 115 extracellular enzyme-producing bacilli were obtained. Casein was hydrolysed by 78%, soluble starch by 67%, citrus pectin by 63%, polygalacturonic acid by 62%, Tween 20 by 34%, birchwood xylan by 16%, Tween 80 by 12%, and carboxymethylcellulose by 3% of the isolates. The isolates were differentiated into 19 distinct homology groups by the 16S-ITS rDNA restriction pattern analysis. Eight different extracellular enzyme activities were determined in 115 endospore forming bacilli. The largest 16S-ITS rDNA homology group (HT1) included 36% of the isolates, 98% of which degraded casein, polygalacturonic acid, pectin and starch. This study is the first report on the characterization of the industrial enzyme-producing alkalophilic bacilli by 16S-ITS rDNA restriction fragment length polymorphism (RFLP). Restriction profiles of 64% of the isolates were found to be different from those of five reference strains used.

Nucleoli, rRNA genes and ITS region in Posidonia oceanica (L.) Delile.

The maximum number of nucleoli was counted in interphase nuclei of Posidonia oceanica, and a restriction pattern of nuclear rDNA was obtained after digestion with four restriction endonucleases and Southern hybridization. P. oceanica has only one type of ribosomal gene whose size was estimated to be 18.5 kbp long. The nucleotide sequence of the entire ITS region was also determined by direct sequencing of PCR amplified DNA fragments. The sequence of the ITS region was aligned with those of homologous regions of other monocots available in literature, and phylogenetic trees were obtained.

On the hybrid origin of Narcissus biflorus (Amaryllidaceae): analysis of C-banding and rDNA structure

Abstract Giemsa and fluorochrome banding with DAPI and chromomycin A3, were utilized to assess karyological details which correlate N. biflorus with the parental species: N. tazetta and N. poeticus. The banding profile in N. biflorus clearly reproduced the model of its progenitors. The EcoR1 restriction pattern of rDNA obtained by Southern blot hybridization indicated, in our material, that each species has more than one ribosomal gene type and in N. biflorus both the ribosomal repeat units of the progenitor species are present.

Molecular chemotaxonomy of Daldinia and other Xylariaceae

In a polyphasic classification approach, stromata and cultures of Daldinia and allied Xylariaceae from around the world were studied for: (1) morphology of teleomorphs and anamorphs; (2) metabolite patterns in stromata and cultures, employing analytical HPLC-UV-visible (diode array) detection and mass spectrometric detection; (3) amplified 18S rDNA restriction patterns (ARDRA); and (4) PCR amplified minisatellite regions. Comparison of type material, herbarium specimens, cultures, and freshly collected material revealed new evidence on their geographic distribution. Several Daldinia spp. were identified in Europe and other locations for the first time. The results point towards the existence of further undescribed species. Stromata of a given species never contained the same major metabolites as corresponding cultures. Most cultures of Daldinia spp. produced naphthalene and chromane derivatives, differing from allied genera by the absence of mellein. Stromata of Daldinia spp. did not produce mitorubrin, but generally contained binaphthyls. Metabolite profiles were correlated with colours of KOH-extractable stromatal pigments. The yellow azaphilones and benzophenones found in D. childiae were lacking in species with purple stromatal pigments. Cytochalasins were found in stromata of D. eschscholzii. Genetic fingerprints helped to distinguish morphologically closely related taxa. ARDRA gave specific results for species whose 18S rDNA contained insertions, while minisatellite PCR provided specific genetic fingerprints. A combination of both PCR based techniques provided a fair resolution of genetic subtypes, reflecting the intrageneric variance in Daldinia as established from morphological data and secondary metabolite profiles.

Host Range of a California Sting Nematode Population

Recently, sting nematodes were discovered associated with dying turfgrass in several golf courses in Coachella Valley, Calif. Based on their morphology and internal transcribed spacer (ITS) rDNA restriction pattern, the pests were identified as Belonolaimus longicaudatus Rau. This study was undertaken to determine the host status of 60 different plant species and cultivars for a California population of B. longicaudatus. The host range tests were conducted under greenhouse conditions at 25 ± 2 °C and ambient light. At the second-leaf stage, each pot was infested with 55 ± 12 adults or fourth-stage juveniles per 150 g of blow sand. The population densities determined after 7 weeks of incubation qualified >80% of the plants tested as good hosts with a reproduction factor (Rf = Pf/Pi) > 4. The majority of those were grasses, although reproduction was best on Gossypium hirsutum L. with Rf = 58.6. While Capsicum annuum L., Medicago sativa L., Arachis hypogaea L., Euphorbia glyptosperma Engelm., Cucumis sativus L., and Daucus carota L. were less suitable host plants with Rf < 4, only Abelmoschus esculentus (L.) Moench, Citrullus lanatus Thunb., and Nicotiana tabacum L. were nonhosts among the tested species. This sting nematode population had a high reproductive fitness on a majority of species tested and must be considered a major threat for most agricultural and horticultural crops grown in sandy soils.

Classification of Brucella strains isolated from marine mammalsusing DNA–DNA hybridization and ribotyping

DNA–DNA hybridization showed that the Brucella strains recently isolated from marine mammals belong to the monospecific genus Brucella (more than 77% DNA relatedness). Ribotyping ( HindIII rDNA restriction patterns) showed that they may represent a separate subgroup (marine type) specifically associated with marine mammals.

New molecular evidence on genome relationships and chromosome identification in fescue (Festuca) and ryegrass (Lolium)

The level of similarity between the DNA of Lolium multiflorum and three Festuca species, F. arundinacea, F. pratensis and F. glaucescens, was analysed on Southern blots, using DNA–DNA hybridization, and also on chromosomes using genomic in situ hybridization (GISH). It was demonstrated that the close relationship between L. multiflorum and the allohexaploid F. arundinacea arises principally from the affinity of L. multiflorum to one of the ancestral progenitors of F. arundinacea, namely F. pratensis. Using probes made from total genomic DNA of L. multiflorum, and also of L. perenne, particular regions of high homology, described here as ‘GISH bands’ were identified on F. pratensis chromosomes. These GISH bands, in combination with a specific probe for rDNA, provide us with new markers for the identification of chromosomes within the F. pratensis complement. Restriction analysis within the rDNA repeat unit revealed additional information on the close phylogeny of L. multiflorum and F. pratensis. The rDNA restriction patterns confirm that F. arundinacea originated as a hybrid between F. pratensis and F. glaucescens.

New molecular evidence on genome relationships and chromosome identification in fescue (Festuca) and ryegrass (Lolium)

The level of similarity between the DNA of Lolium multiflorum and three Festuca species, F. arundinacea, F. pratensis and F. glaucescens, was analysed on Southern blots, using DNA–DNA hybridization, and also on chromosomes using genomic in situ hybridization (GISH). It was demonstrated that the close relationship between L. multiflorum and the allohexaploid F. arundinacea arises principally from the affinity of L. multiflorum to one of the ancestral progenitors of F. arundinacea, namely F. pratensis. Using probes made from total genomic DNA of L. multiflorum, and also of L. perenne, particular regions of high homology, described here as ‘GISH bands’ were identified on F. pratensis chromosomes. These GISH bands, in combination with a specific probe for rDNA, provide us with new markers for the identification of chromosomes within the F. pratensis complement. Restriction analysis within the rDNA repeat unit revealed additional information on the close phylogeny of L. multiflorum and F. pratensis. The rDNA restriction patterns confirm that F. arundinacea originated as a hybrid between F. pratensis and F. glaucescens.

Diversity of denitrifying microflora and ability to reduce N 2 O in two soils

The ozone-depleting gas N2O is an intermediate in denitrification, the biological reduction of NO3 – to the gaseous products N2O and N2 gas. The molar ratio of N2O produced (N2O/N2O+N2) varies temporally and spatially, and in some soils N2O may be the dominant end product of denitrification. The fraction of NO3 –-N emitted as N2O may be due at least in part to the abundance and activity of denitrifying bacteria which possess N2O reductase. In this study, we enumerated NO3 –-reducing and denitrifying bacteria, and compared and contrasted collections of denitrifying bacteria isolated from two agricultural soils, one (Auxonne, soil A) with N2O as the dominant product of denitrification, the other (Châlons, soil C) with N2 gas as the dominant product. Isolates were tested for the ability to reduce N2O, and the presence of the N2O reductase (nosZ)-like gene was evaluated by polymerase chain reaction (PCR) using specific primers coupled with DNA hybridization using a specific probe. The diversity and phylogenetic relationships of members of the collections were established by PCR/restriction fragment length polymorphism of 16s rDNA. The two soils had similar numbers of bacteria which used NO3 – as a terminal electron acceptor anaerobically. However, the soil A had many more denitrifiers which reduced NO3 – to gaseous products (N2O or N2) than did soil C. Collections of 258 and 281 bacteria able to grow anaerobically in the presence of NO3 – were isolated from soil A and soil C, respectively. These two collections contained 66 and 12 denitrifying isolates, respectively, the others reducing NO3 – only as far as NO2 –. The presence of nosZ sequences was generally a poor predictor of N2O reducing ability: there was agreement between the occurrence of nosZ sequences and the N2O reducing ability for only 42% of the isolates; 35% of the isolates (found exclusively in soil A) without detectable nosZ sequences reduced N2O whereas 21% of the isolates carrying nosZ sequences did not reduce this gas under our assay conditions. Twenty-eight different 16S rDNA restriction patterns (using two restriction endonucleases) were distinguished among the 78 denitrifying isolates. Two types of patterns appeared to be common to both soils. Twenty-three and three types of patterns were found exclusively among bacteria isolated from soils A and C, respectively. The specific composition of denitrifying communities appeared to be different between the two soils studied. This may partly explain the differences in the behaviour of the soils concerning N2O reduction during denitrification.

Genotypic Characterization of Bradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata.

Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S Ribosomal DNA for Rapid Identification of Saccharomonospora Strains

Twenty-one strains of Saccharomonospora azurea, Saccharomonospora cyanea, Saccharomonospora glauca, Saccharomonospora viridis, and “Saccharomonospora caesia” were examined to evaluate the discriminatory value of 16S ribosomal DNA (rDNA) fingerprints. The 16S rDNAs were amplified by PCR by using oligonucleotide primers complementary to 16S rRNA genes. A restriction fragment length polymorphism (RFLP) analysis of the 16S rDNAs was performed with SmaI and MluI. The four validly described Saccharomonospora species could be differentiated on the basis of their characteristic 16S rDNA restriction patterns. The strains of “S. caesia” gave a restriction pattern identical to that of S. azurea K161T (T = type strain). This result was anticipated from the previous report that S. azurea K161T and the strains of “5. caesia” have identical 16S rRNA sequences. We found that purification of amplified 16S rDNA products following PCR was necessary for our RFLP analysis.

Phylogenetic characterization of a new thermoacidophilic bacterium isolated from hot springs in Japan.

Three strains of thermophilic-acidophilic bacteria isolated previously from different hot springs in Japan were characterized by molecular genetic methods. The strategy taken involved PCR amplification, sequencing and restriction pattern analysis of 16S rDNA, 16S-23S rDNA spacer polymorphism analysis and genomic DNA-DNA hybridization. A phylogenetic analysis based on 16S rDNA sequences showed that the new thermoacidophilic isolates formed a genetically coherent group at the species level and fell into a major cluster together with members of the genera Alicyclobacillus and Sulfobacillus with A. acidocaldarius and A. acidoterrestris as their closest relatives. The levels of binary sequence similarity between the isolates and the two Alicyclobacillus species were 97.6 to 97.9%, values considered low enough to warrant placement of the isolates in a distinct species of the genus Alicyclobacillus. The 16S rDNA restriction pattern analysis, but not 16S-23S rDNA spacer polymorphism analysis, was useful for differentiating the isolates from the established Alicyclobacillus species. DNA-DNA hybridization assays demonstrated a distinct phylogenetic position of our isolates as a genospecies within the genus Alicyclobacillus. On the basis of these results, the thermoacidophilic isolates should be classified into a new species of Alicyclobacillus. The results of this study suggest that this new genospecies of Alicyclobacillus is widely distributed in hot springs in Japan.

Rapid Distinction of Brevibacterium Species by Restriction Analysis of rDNA Generated by Polymerase Chain Reaction

Summary Twelve reference strains, including the type strains, of species either belonging or related to the genus Brevibacterium , and nineteen well-characterized clinical isolates assigned to this genus were studied. Polymerase chain reaction was used for partial amplification of the gene coding for their 16S rRNA (rDNA genes) which were digested with nine different restriction endonucleases. The amplified rDNAs were approximately 1300 bp long. The type strains of B. casei and B. epidermidis exhibited different Bst NI rDNA restriction patterns. Furthermore, these patterns differed from those observed for B. linens and B. iodinum . Of the clinical isolates fifteen had the same BstNl restriction pattern as the B. casei type strain, two the same as the B. epidermidis type strain, one the same as B. linens , and one strain had a particular pattern. Results obtained by rDNA restriction analysis were confirmed by DNA-DNA hybridization studies, which revealed that representatives of the fifteen isolates resembling B. casei shared more than 75% homology with B. casei CIP102111 T . Combined with a rapid DNA extraction procedure, PCR-amplified-rDNA restriction fragment analysis allowed the distinction of the different Brevibacterium species in less than seven hours. The results suggested that most brevibacteria from clinical samples belongs to B. casei rather than B. epidermidis , as commonly thought. This approach may also be of value for differentiation within other genera of Gram-positive bacteria.