Biofouling on the walls of a spent nuclear fuel pool with radioactive ultrapure water

Microbial activity in spent nuclear fuel pools which contain ultrapure and radioactive water has been previously observed. The aim of the present research was to isolate and identify the microorganisms attached to the nuclear pool wall of a Spanish nuclear power plant. Amplification of 16S rDNA fragments from the culturable microorganisms by PCR using universal primers for the domain 'Bacteria', followed by Denaturing Gradient Gel Electrophoresis analysis revealed the presence of six different bacteria. The complete gene for 16S rDNA of each one was sequenced and identified as belonging to three different phylogenetic groups, viz. beta-Proteobacteria, Actinomycetales and the Bacillus/Staphylococcus group. A fungus was also found and identified as Aspergillus fumigatus by sequencing the D2 region of the large subunit rDNA gene. The isolation of these microorganisms in oligotrophic and radioactive conditions is of great interest due to the possibility of their use in bioremediation processes of radionuclide-contaminated environments.     

Isolation and identification of bacteria from spent nuclear fuel pools

The aim of the present research was to isolate and identify bacteria from spent nuclear fuel pools of a Spanish nuclear power plant. Water samples were collected and inoculated onto different culture media to isolate the highest number of species. 16S rDNA fragments from colonies growing on solid media were amplified and analysed by denaturing gradient gel electrophoresis. Sequencing revealed the presence of 21 different bacteria belonging to several phylogenetic groups (, , and -Proteobacteria, Actinomycetales, Flavobacterium, and the Bacillus/Staphylococcus group). The isolation of these microorganisms in this particular environment (oligotrophic and radioactive) is highly interesting because of the possibility of their being used for the bioremediation of radionuclide-contaminated waters.     

Identification of Mytilus spp. and Pecten maximus in Irish Waters by Standard PCR of the 18S rDNA Gene and Multiplex PCR of the 16S rDNA Gene

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.     

Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (=Gymnodinium chlorophorum)

The ultrastructure of the green dinoflagellate Lepididodinium viride M. M. Watanabe, S. Suda, I. Inouye Sawaguchi et Chihara was studied in detail. The nuclear envelope possessed numerous chambers each furnished with a nuclear pore, a similar arrangement to that found in other gymnodinioids. The flagellar apparatus was essentially identical to Gymnodinium chlorophorum Elbrächter et Schnepf, a species also containing chloroplasts of chlorophyte origin. Of particular interest was the connection of the flagellar apparatus to the nuclear envelope by means of both a fiber and a microtubular extension of the R3 flagellar root. This feature has not been found in other dinoflagellates and suggests a close relationship between these two species. This was confirmed by phylogenetic analysis based on partial sequences of the large subunit (LSU) rDNA gene of L. viride, G. chlorophorum and 16 other unarmoured dinoflagellates, including both the ‘type’ culture and a new Tasmanian isolate of G. chlorophorum. These two isolates had identical sequences and differed from L. viride by only 3.75% of their partial LSU sequences, considerably less than the difference between other Gymnodinium species. Therefore, based on ultrastructure, pigments and partial LSU rDNA sequences, the genus Lepidodinium was emended to encompass L. chlorophorum comb. nov.     

Phylogenetic analysis of the insect order Odonata using 28S and 16S rDNA sequences: a comparison between data sets with different evolutionary rates

Molecular phylogenetic analyses were conducted for the insect order Odonata with a focus on testing the effectiveness of a slowly evolving gene to resolve deep branching and also to examine: (i) the monophyly of damselflies (the suborder Zygoptera); and (ii) the phylogenetic position of the relict dragonfly Epiophlebia superstes. Two independent molecular sources were used to reconstruct phylogeny: the 16S rRNA gene on the mitochondrial genome and the 28S rRNA gene on the nuclear genome. A comparison of the sequences showed that the obtained 28S rDNA sequences have evolved at a much slower rate than the 16S rDNA, and that the former is better than the latter for resolving deep branching in the Odonata. Both molecular sources indicated that the Zygoptera are paraphyletic, and when a reasonable weighting for among‐site rate variation was enforced for the 16S rDNA data set, E. superstes was placed between the two remaining major suborders, namely, Zygoptera and Anisoptera (dragonflies). Character reconstruction analysis suggests that multiple hits at the rapidly evolving sites in the 16S rDNA degenerated the phylogenetic signals of the data set.     

Identification of Arcanobacterium haemolyticum isolated from postcastrational complications of a horse

An Arcanobacterium haemolyticum strain isolated from a postcastrational lesion of a horse was identified phenotypically and genotypically. The latter was performed by sequencing the 16S–23S rDNA intergenic spacer region (ISR), by amplification of the gene encoding A. haemolyticum phospholipase D, by amplification of A. haemolyticum specific parts of ISR-23S rDNA and by amplification of the newly described CAMP factor family protein encoding gene of A. haemolyticum. This indicates (as described previously for seven additional A. haemolyticum strains; Hassan et al. 2009) that A. haemolyticum seems to occur also in infections of horses.     

Evaluation of PCR Based on Gene <Emphasis Type="Italic">apxIVA</Emphasis> Associated with 16S rDNA Sequencing for the Identification of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> and Related Species

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.     

Characterization of Astragalus sinicus Rhizobia by Restriction Fragment Length Polymorphism Analysis of Chromosomal and Nodulation Genes Regions

Two hundred and four isolates of rhizobia were sampled from root nodules of Astragalus sinicus grown in rice fields of six southern provinces of China. Genotypic diversity was determined by Southern hybridization using nodDBC genes as a probe, restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S-23S rDNA intergenic spacers (IGS), and plasmid profile. Our results show that rhizobia associated with A. sinicus were very diverse, and 10 genotypes were resolved within the previously identified dominant 16S rDNA type. Diversity levels varied greatly between different geographical locations. The same nod gene genotypes were harbored by distinct chromosomal types, suggesting that lateral plasmid transfer occurred during the evolution process. Received: 14 June 1999 / Accepted: 20 July 1999     

Phylogenetic relationships in the millipede family Julidae

A phylogenetic analysis of 40 species (22 genera) of the Palaearctic millipede family Julidae was made based on partial sequences of the mitochondrial 16S rRNA (16S) gene and the nuclear 28S rRNA (28S) gene, respectively. The two data sets (16S rDNA and 28S rDNA) were analysed individually and in combination using direct optimization as implemented in POY. The 16S rDNA and the 28S rDNA sequences vary from 410 to 449 bp and from 467 to 525 bp in length, respectively. All searches were performed under six different gap opening costs, an extension gap cost of 1, and a substitution cost of 2. Incongruence length difference values were used to select the preferred tree. The order Julida was recovered as monophyletic under all weight sets. The family Julidae was recovered as monophyletic except under one weight set where the genus Nepalmatoiulus is sister to all other Julida. Within Julidae, a clade of Paectophyllini + Calyptophyllini is sister to all others on the preferred tree but this relationship is not robust. A hitherto unrecognized clade of (South) east Asian genera (Anaulaciulus and Nepalmatoiulus) was recovered under five weight sets. Another “new” robust clade (Oncoiulini + Schizophyllini) is congruent with a hitherto unrecognized complex morphological character. Further clades recovered within the Julidae partly conflict with the accepted classification, which is only to a limited extent based on phylogenetic arguments.     

Development and characterization of biofilms on stainless steel and titanium in spent nuclear fuel pools

The aim of the present research was to study the biofilms developed in a Spanish nuclear power plant and their ability to entrap radionuclides. In order to carry this out, a bioreactor, which was then submerged in a spent nuclear fuel pool, was designed. To characterise the biofilm on two different metallic materials (stainless steel and titanium), standard culture microbiological methods and molecular biology tools, as well as epifluorescence and scanning electron microscopy were used. The bacterial composition of the biofilm belongs to several phylogenetic groups (α, β, and γ-Proteobacteria, Actinobacteridae, and Firmicutes). The radioactivity of the biofilms was measured by gamma-ray spectrometry. Biofilms were able to retain radionuclides from radioactive water, especially ⁶⁰Co. The potential use of these biofilms in bioremediation of radioactive water is discussed.     

Identification of ‘Candidatus Phytoplasma asteris' (16SrI‐B) Causing Witches' Broom Disease of Mallotus japonicus in Korea

Mallotus japonicus with witches' broom disease were observed in Jeollabuk‐do, Korea. A phytoplasma from the infected leaves was identified, based on the 16S rDNA, 16S‐23S intergenic spacer region, and fragment of rp operon and tuf gene sequences. The 16S rDNA sequences exhibited maximum (99.7%) similarity with Iranian lettuce phytoplasma, the rp operon sequences exhibited 100% similarity with Goldenrain stunt phytoplasma, and the tuf gene sequences exhibited 99.8% similarity with Japanese spurge yellows phytoplasma. Results of the sequence analysis and phylogenetic studies confirmed that the phytoplasma associated with M. japonicus in Korea was an isolate of Aster Yellows group (subgroup16SrI‐B).     

Electrical DNA-chip-based identification of different species of the genus Kitasatospora

The identification of different Kitasatospora strains has been shown with a DNA-chip based on an electrical readout scheme. The 16S-23S rDNA internal transcribed spacer region of these Actinomycetes was used for identification. Two different capture probes per strain were immobilized on the chip. The capture probes were spotted on a DNA-chip with electrode structures for an electrical DNA detection. A biotinylated PCR product of the 16S-23S rDNA region was incubated on the chips and bound to its complementary capture sequences. Followed by a gold nanoparticle or enzyme labeling and a deposition of silver, the binding of the PCR product was detected by an increase of the measured conductivity on the chip. To show the applicability of this detection system, four strains of Kitasatospora were chosen for an identification using the DNA-chip with electrical detection. Each strain was clearly identified using the system. Concentrations of the polymerase chain reaction (PCR) products within the range of 1 ng/ml to 1 μg/ml were detected and identified. These tests are the first application of this novel electrical detection scheme for the identification and classification of microorganisms. The presented results show that the DNA-chip with electrical detection can be used for a robust and cost-efficient DNA analysis.     

Microbial Diversity during Biodegradation of Crude Oil in Seawater from the North Sea

Microbial communities were characterized during biodegradation of immobilized oil in seawater from the Statfjord field and the German Bight in the North Sea. Seawater samples were collected at different distances from pollution sources at the two locations. A Statfjord oil was immobilized on hydrophobic synthetic Fluortex fabrics and submerged in closed flasks (no headspace) with natural or sterile seawater and incubated at 13°C for 56 days. Biodegradation of immobilized n-alkanes was measured by gas chromatography, total microbes were enumerated by epifluorescence microscopy, and culturable heterotrophic and oil-degrading microorganisms were quantified by most probable number (MPN) analysis. Polymerase chain reaction (PCR) amplification of bacterial 16S rDNA in water samples was conducted during biodegradation experiments. The amplified 16S rDNA fragments were characterized by denaturing gradient gel electrophoresis (DGGE), and by sequence analysis of cloned inserts. Biodegradation rates of alkanes in seawater collected at different distances from the pollution sources did not differ significantly (P > 0.05). Concentrations of oil-degrading microorganisms showed a temporary peak after 7 days of degradation, with a subsequent decline later in the period. DGGE analysis of 16S rRNA genes showed that community diversity decreased during the first 2–3 weeks of biodegradation, with the emergence of a few dominant bands. Cloning, restriction analysis, and sequence analysis of the 16S rDNA fragments revealed >30 different phylotypes. Abundant types during biodegradation belonged to the -Proteobacteria, in waters from both Statfjord and the German Bight. Cloning and sequencing studies indicated that the most abundant bacteria during biodegradation belonged to the family Rhodobacteraceae, with the closest relationship to the genera Sulfitobacter and Roseobacter.     

Phylogenetic analysis of the fecal flora of the wild pygmy loris

The bacterial diversity in fecal samples from the wild pygmy loris was examined with a 16S rDNA clone library and restriction fragment length polymorphism analysis. The clones were classified as Firmicutes (43.1%), Proteobacteria (34.5%), Actinobacteria (5.2%), and Bacteroidetes (17.2%). The 58 different kinds of 16S rDNA sequences were classified into 16 genera and 20 uncultured bacteria. According to phylogenetic analysis, the major genera within the Proteobacteria was Pseudomonas, comprising 13.79% of the analyzed clone sequences. Many of the isolated rDNA sequences did not correspond to known microorganisms, but had high homology to uncultured clones found in human feces. Am. J. Primatol. 72:699–706, 2010. © 2010 Wiley‐Liss, Inc.     

Genetic and chaetal variation in Nais worms (Annelida,Clitellata, Naididae)

The genus Nais is a group of oligochaetous clitellates, common in eutrophic freshwater habitats. About 30 species are described. Species identification is based primarily on chaetal characters, which are often subtle, inconsistent, and even overlapping between nominal species. We investigated the correlation between genetic variation and chaetal morphology in this genus. Eighty‐one individuals from Europe, North America, and China were included in the study. Seventy‐five of these were preserved as vouchers. They were scrutinized with regard to chaetal morphology, and ten different morphotypes were identified. Three molecular markers, two mitochondrial (the COI gene and 16S rDNA) and one nuclear (the ITS region), were used to establish the genetic lineages in the material. Genetic variation was found to be largely congruent with chaetal character patterns. However, at least nine separately evolving lineages (all supported by mitochondrial as well as nuclear data) correspond to at most six nominal species. Four morphotypes/lineages are recognized as Nais barbata, Nais christinae, Nais elinguis, and Nais stolci, respectively, whereas five, or possibly more, lineages represent a morphological continuum covering the variation of the Nais communis/variabilis complex. Thus, cryptic speciation is revealed. Our results indicate that a taxonomic revision of the genus will be needed in the future.     

Molecular Ecological Analysis of Methanogens and Methanotrophs in Blanket Bog Peat

Abstract Methane production and methane oxidation potential were measured in a 30 cm peat core from the Moorhouse Nature Reserve, UK. The distribution of known groups of methanogens and methane oxidizing bacteria throughout this peat core was assessed. Using 16S rRNA gene retrieval and functional gene probing with genes encoding key proteins in methane oxidation and methanogenesis, several major groups of microorganisms were detected. Methane production and oxidation was detected in all depths of the peat core. PCR amplification and oligonucleotide probing experiments using DNA isolated from all sections of the peat core detected methanotrophs from the groups Methylosinus and Methylococcus and methanogens from the groups Methanosarcinaceae, Methanococcaceae, and Methanobacteriaceae. 16S rDNA sequences amplified with the Methylosinus-specific primer were shown to have a high degree of identity with 16S rDNA sequences previously detected in acidic environments. However, no methanogen sequences were detected by the probes available in this study in the sections of the peat core (above 7 cm) where the majority of methanogenesis occurred, either because of low methanogen numbers or because of the presence of novel methanogen sequences. Received: 9 March 1999; Accepted: 21 June 1999     

Species composition of the association of acidophilic chemolithotrophic microorganisms participating in the oxidation of gold-arsenic ore concentrate

The species composition of the microbial association involved in industrial tank biooxidation of the concentrate of refractory pyrrhotite-containing pyrite-arsenopyrite gold-arsenic ore of the Olympiadinskoe deposit at 39°C was studied by cultural and molecular biological techniques. Pure microbial cultures were isolated, their physiological characteristics were investigated, and their taxonomic position was determined by 16S rRNA gene sequencing. The library of 16S rRNA gene clones obtained from the total DNA isolated from the biomass of the pulp of industrial reactors was analyzed. The diversity of microorganisms revealed by cultural techniques in the association of acidophilic chemolithotrophs (Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, Ferroplasma acidiphilum, Alicyclobacillus tolerans, and Acidiphilium cryptum) was higher than the diversity of the 16S rDNA clone library (At. ferrooxidans, L. ferriphilum, and F. acidiphilum). The combination of microbiological and molecular biological techniques for the investigation of the biodiversity in natural and anthropogenic microbial communities promotes detection of new phylogenetic microbial groups in these communities.     

佳西热带雨林土壤芽胞杆菌分离与多样性分析

采用热处理法从海南省佳西热带雨林土壤中分离到147株芽胞杆菌,并利用16S rDNA PCR-RFLP与序列分析技术对其遗传多样性进行了研究。16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析结果表明,在100%的相似性水平上,这些芽胞杆菌分属13个遗传类群。不同遗传类型代表菌株的16S rRNA基因序列分析结果显示,它们分布在Bacillaceae、Planococcaceae和Paenibacillaceae科的Bacillus、Lysinibacillus、Paucisalibacillus、Bhargavaea和Paenibacillus五个属,其中Bacillus为优势属(占50%);有3株芽胞杆菌的16S rRNA基因序列与数据库中相应模式菌株的最大相似性在98.3%~98.9%之间。结果表明,佳西热带雨林土壤中芽胞杆菌有着较为丰富的遗传多样性。     

Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.     

Comparison between the organization of nuclear ribosomal DNA unit of Euglena gracilis Z and var. Bacillaris

Summary We have characterized the nuclear rDNA unit of Euglena gracilis var. bacillaris and compared it to that of the Z strain. We have localized restriction sites for Eco R1, Sal 1, Sma 1, Hind III, Bam H1 and Bgl II on this unit as well as the coding region for 20 S and 25 S rRNAs. For both strains, results suggest an homogeneity of the 11.6 kbp rDNA units. Comparison between strains shows differences characterized by two additional Sal 1 sites in bacillaris and the likely methylation of one Sma 1 site in Z. Both differences are localized in a non-coding region of the rDNA unit. Analyses of 18 Euglena strains from various origins confirm these differences and allow easy recognition of bacillaris and Z type strains.Abbreviations kb kilo base - kpb kilo base pair - plasmids pRH 59 and pRH 57 contain a Hind III-HInd III nuclear DNA fragment from W₃BUL of 5.9 and 5.7 kbp respectively, pRB 48 and pRB 35 contain a Bam H1-Bam H1 nuclear DNA fragment from wild-type Z of 4.8 and 3.5 kbp respectively - SDS sodium dodecyl sulfate - UV ultra-violet